Some Experimental Protocols Used:

G.R.Kantharaj

MATERIALS AND METHODS

Materials:

Seeds of Phaseolus vulgaris. L. have been procured from Lalbagh seed Nursery center, Bangalore , India. [¹⁴C] Chlorella protein hydrolysate (27 Ci/m atom carbon,) and [³²p] orthophosphate (carrier-free) were purchased from Babha atomic Research center, Bombay, India. [³H]-Leucine (105 Ci/mmole) and [³H]-Uridine (27 Ci/mmole) were purchased from Radiochemical Centre, Amersham, Bucks, U.K. Oligo dT-cellulose was purchased from Collaborative Research Inc., Waltham, Mssachussetts, U.S.A. Poly (U)-sepharose was purchased from sigma Chemicals co., St. Louis, U.S.A. All other chemicals used were of Analytical Grade.

Methods:

Morphological Studies:

Phaseolus vulgaris L. seedlings were raised in sand, in a dark chamber. When the hypocotyls attained a height of 15-20 cm, they were cut at the base. Hypocotyls of 8-10 cm length were washed in tap water for 30 minutes and kept in required volume of half strength Hoagland’s nutrient solution [220], with or without plant hormones. In all long-term experiments a 12 hr day period was maintained using fresh medium everyday. To prevent bacterial contamination, 30 hg /ml of Ampicillin (Hindustan Antibiotics Ltd.) was added to the culture medium. The number of roots produced was counted at the end of 4^th day.

For anatomical studies, 1 cm length hypocotyls segments were excised and fixed in Conroy’s solution (70 ml of 70% ethanol, 20 ml chloroform and 10 ml of glacial acetic acid). Then the segments were embedded in paraffin wax. Microtome sections (10 m thickness) were taken and processed as described by Jensen (1962) [230]. Microphotographs were taken using 100 ASA panchromatic film.

Protein synthesis in vivo

Protein synthesis in excised hypocotyls was followed by placing the required amount of tissue (suitably treated and washed) in a known amount of half strength Hoagland’s solution containing the required amount of [¹⁴ C] – chlorella protein hydrolysate. The different treatments given, the amount of tissue and the label used are described in the legends of respective tables and figures.

The labeled segments were thoroughly washed with distilled water to remove any adhering radioactivity and the tissue homogenate was prepared by grinding in O.o2M sodium phosphate buffer (pH 7.0) in the cold (0-4^o). Perchloric acid was added to the homogenate to a final concentration of 0.3M (v/v) and the precipitate recovered by centrifugation after standing for a few hours in cold. The protein was extracted from the precipitate in 1N NaOH, neutralized and reprecipitated by adding 50% trichloroacetic acid (TCA) to a final concentration of 10% (v/v). The precipitate was successively washed with 10% hot TCA (90^oC), 10% cold TCA, ethanol-ether (3:1, v/v) and finally with ether. The final preparation, after the removal of ether was dissolved in formic acid and aliquots were planchetted on Whatman No.3 filter paper discs, dried and used for radioactivity measurements. Similar aliquots, after neutralization, were also used for protein estimation by the method of Lowry et al[221].

The specific radioactivity of the amino acid pool was estimated using the 0.3M perchloric acid supernatant. The supernatant was neutralized and aliquots were used for radioactivity measurements. Total amino acid content was estimated by the method of Rosen [222].

Protein synthesis in vitro

The procedure used for the isolation of polysomes from the hypocotyls segments were as described by Anderson et al. [223]. The tissue was homogenized in 5 volumes of buffer a (containing 50 mM Tris-HC1 (pH 7.5), 5 mM MgC1₂, 15 mM KCL, 0.6 mM DTT and 300 mM sucrose). The homogenate was centrifuged at 20000xg for 15 min and 2 ml of the supernatant was layered over 3ml of 1.5M sucrose pad and centrifuged at 150000xg for 2 hr. The pellet was suspended in the homogenizing buffer A. Another batch of homogenate was centrifuged at 20000xg for 15 min and the supernatant was centrifuged at 150000xg for 2 hr directly without layering on sucrose. The final supernatant was passed through a G-25 Sephadex column (20xl.5cm) which was already equilibrated with homogenizing buffer. Fractions with maximum A₂₈₀values were pooled and used as the source of soluble enzymes (8-150 supernatant fractions).

The in vitro protein synthesizing incubation mixture in a total volume of 100 m1 contained 20mm HEPES buffer (pH 7.5), 5 mM MgCl_2,50 mM KCL, 0.4 mM ATP, 0.12 mM GTP, 5 mM phosphoenol pyruvate, 2 enzyme units of pyruvate kinase, 25 mM each of a mixture of amino acid minus leucine, 100 mg RNA from polyribosome, 100mg protein of S-150 supernatant fraction and 1 mCi of [³H] – leucine (105 Ci/mmole). After incubating the mixture for 30 min at 37^oC, aliquots (20 m1) were applied on to filter paper discs. After air-drying, the discs were successively washed with hot TCA (10%) and cold TCA (5%), ethanol –ether (3:1) and finally with ether. The discs were then counted for radioactivity.

RNA Synthesis:

The hypocotyls tissue, after hormone treatment, was incubated for 30min in half strength Hoagland’s nutrient medium containing either carrier free [³² P] – orthophosphate or [³H] –uridine. After incubation the tissue was washed thoroughly with water, then total RNA was extracted by phenol-chloroform procedure [224] with minor modifications.

Isolation of total RNA:

In these experiments, in order to eliminate RNase contamination, all glass wares were washed in acid and baked overnight in hot air oven (200^oC). All aqueous solutions were autoclaved.

The required amount of hypocotyls tissue was homogenized in equal volumes of 0.1M Tris-acetate (pH 9.2) buffer (containing 1% SDS, 0.001M EDTA, 0.1% diethylpyrocarbonate) in a pre-cooled all-glass mortar and pestle. To the homogenate equal volumes of phenol (saturate with buffer) – chloroform (1:1, v/v) was added. After constant stirring for 20-30 min, the extract was centrifuged at 10000kg for 10 min. The aqueous phase was carefully transferred to a separate flask. The interphase was repeatedly extracted with the addition of homogenizing buffer. The aqueous phases were all pooled and equal volumes of phenol-chloroform was added and centrifuged at 10,000kg for 10 min. The aqueous phase was removed and sodium chloride was added to a final concentration of 0.15M. RNA was precipitated with the addition of 2.5 volumes of ethanol and the mixture was left overnight at – 20^oC. The pellet was collected by centrifugation at 15,000 kg for 10 min. The total RNA thus obtained was washed successively with 2 volumes of 70% ethanol containing 2M potassium acetate, and thrice with 3M sodium acetate (pH 6.8) and finally reprecipitated with ethanol.

Isolationof poly (A) – containing RNA:

Poly (A)-containing RNA was isolated by the methods of Padmanabhan et al. (1975) [225]. Total RNA was suspended is 0.01M Tris-HC1 (pH 7.5) buffer containing 0.5M sodium chloride and 0.5% SDS (high-salt buffer). About 250 A ₂₆₀units of total RNA was loaded on to 2.5 ml of poly (U) – Sepharose (0.5 gms) or oligo dT-cellulose (0.5 gm) column equilibrated with high – salt buffer. The flow rate was adjusted to 1 ml/min. The column was first washed with 10ml of high – salt buffer without SDS. Poly (A) containing RNA was eluted with o.o1M Tris-HC1 (pH 7.5) buffer. The poly (A) – RNA thus obtained was subjected to another cycle of affinity chromatography. To the eluates potassium acetate was added to 0.15M concentrations and poly (A)–RNA was precipitated with the addition of 2.5 vol of ethanol. After keeping the RNA mixture for 16-20 hrs at – 20^oC, Poly (A)-RNA was collected by centrifuging at 27000xg for 15 min.

Determination of specific activity of RNA:

In some experiments labeled precursor incorporation into RNA was also followed by measuring the radioactivity released after 0.3M KOH hydrolysis of the 0.3M perchloric acid precipitate obtained with the homogenate. For measuring specific radioactivity of the free nucleotide pool, the supernatant obtained after precipitation of the homogenate with 0.3M HC10₄ was treated with activated charcoal. The charcoal pellet was washed repeatedly with water and finally the nucleotides were eluted with 50% ethanol containing 0.3M NH₄OH [226]. Aliquots were used for measuring A₂₆₀as well as radioactivity.

Analysis of the buffer-extractable proteins labeled in vivo and in vitroPreparation of the buffer-extractable protein fraction labeled in vivo:

Hormone treated hypocotyls tissue (1 gm) was labeled by incubating in 2 ml of Hoaglands nutrients medium containing 25 mCi of [¹⁴ C] – chlorella protein hydrolysate for 30 min. Then the tissue was homogenized in a buffer containing 0.02M sodium phosphate (pH 7.0), 0.25M sucrose and 10mM PMSF. The homogenate was centrifuged at 10,000 rpm for 10 minutes. The supernatant proteins (250 mg) were analyzed by SDS-gel electrophoresis and the radioactivity profile of the separated proteins determined.

Preparation of the protein fraction labeled in vitro:

The labeled proteins synthesized in the homologous cell-free system described were precipitated with 10% TCA. After standing for few hours, the precipitate was collected by centrifugation and washed successively with 10% cold TCA, ethanol-ether (v/v, 3:1) and finally with ether. The dried pellet was used for SDS –gel analysis and measurement of radioactivity profile of the separated proteins.

SDS-Gel electrophoresis:

SDS-polyacrylamide slab gel (8%) electrophoresis was carried out according to Lammli’s procedure [227]. Tris-glycine (pH 8.8) buffer containing 0.1% SDS was used as the electrophoresis buffer. The protein samples were taken in the dissociating buffer containing 10 mM Tris-HC1 (pH 6.8), 10% glycerol, 2% SDS, 5% (v/v) b-mercaptoethanol and heated in boiling water (100^oC) for 2 minutes. The protein samples were loaded into the slots and electrophoresis was carried out at 20 mA constant current (0-4^oC) till the marker dye bromophenol blue reached the bottom of the gel. The gels were removed and fixed in a solution containing 10% acetic acid and 40% methanol for 30min, stained with 0.5 % (w/v) Coomassie brilliant blue-R (Sigma) overnight and destained with the same fixative solution. After destaining, the gels were photographed using red filter. The gels were cut into 1mm slices and digested with H₂ O₂(30%) at 60⁰C for the purpose of radioactivity measurements.

Radioactivity Measurements:

The H₂O₂ digested gel slices were counted using Triton-toluene (1:2, v/v) containing 0.5% PPO in a Beckman LS-100 scintillating counter. Filter discs were counted using 0.5% PPO in toluene as the scintillant. Under the conditions used ³H, ¹⁴C and ³²P were counted with efficiencies of 6%, 35% and 80% respectively.

MATERIALS AND METHODS

Materials:

[³H] – Leucine (105 Ci / mmole) was purchased from Radiochemical Centre, Amersham, bucks, England. Oligo-dT-cellulose was purchased from Collaborative Research Inc., Waltham, Massachusetts, U.S.A. All other chemicals used were of analytical grade.

Methods:

Treatment of the hypocotyls :

As described in Chapter I, hypocotyls segments were placed in Hoagland’s medium containing IBA (30 mg/ml). After 30 min exposure, the hypocotyls were thoroughly washed with water and transferred to flasks containing fresh hormone-free medium. The hypocotyls were removed at different intervals of time and used for RNA isolation.

Isolation of total and poly(A) – containing RNA:

The methods for isolation of total RNA as well as poly(A)-RNA have already been described in Chapter I. Total RNA isolated from 50-100 fm of hypocotyls tissue was washed with 2 volumes of 70% ethanol containing 2M Potassium acetate and then thrice with 3M sodium acetate (pH 6.8). The RNA was reprecipitated with ethanol and the pellet was used for translation. Poly(A)-containing RNA was isolated using two cycles of Oligo-dT-cellulose chromatography.

Translation of RNA in the wheat germ cell-free system:

The procedure of Roberts and Patterson [234] was used with some modifications. In these experiments glassware was washed in acid and baked overnight at 200^oC and all aqueous solutions were autoclaved in order to eliminate inadvertent RNase contamination.

Preparation of S-30 extract:

Wheat germ (1 gm) was ground with 4 ml of buffer A (containing 200 mM HEPES, pH 7.5, 1 mM magnesium acetate, 100mM KC1, 2mM CaCl₂ and 6 mM b-mercaptoethanol) and 1 gm sand in a pre-cooled mortar and pestle. The homogenate was centrifuged at 15000xg for 10 minutes. The supernatant (1.5 ml) was fed onto a Sephadex G-25 column equilibrated with buffer B (containing 20 mM HEPES, pH 7.5, 5 mM magnesium acetate, 120 mM KCl and 6 mM b-mercaptoethanol). The void volume was collected and peak fractions (A₂₆₀) were pooled and centrifuged at 15000xg for 10 minutes. The supernatant was used as the S-30 source.

Assay procedure :

The assay mixture in a total volume of 0.1 ml contained : HEPES, 20 mM; ATP, 1 mM; GTP, 20 mM; Phospho creatine, 8 mM; magnesium acetate, 3 mM; potassium chloride, 100 mM; amino acid mixture without Leucine, 25mM each; DTT, 2 mM; [³H] – Leucine 105 Ci / mmol), 5 mCi; S-30 fraction, about one A₂₆₀unit and RNA (optimum concentration). The mixture was incubated at 25^oC for 1 hr and 5 ml aliquots were transferred to filter paper discs to be processed for measurement of radioactivity in the TCA perceptible proteins as described in experimental methods of Chapter I

SDS-Gel analysis of labeled proteins synthesized in the cel-free system derived from the wheat – germ:

The labeled protein products synthesized in the wheat germ cell-free system in response to RNA isolated from hypocotyls with and without hormone treatment, were precipitated with cold trichloroacetic acid (10% in final concentration) and then washed successively with cold TCA (5%), ethanol-ether (3:1), and finally with ether. The pellet was dissociated in 2% SDS containing 5% b-mercaptoethanol, 10mM Tris – HCL (pH 6.8) and 10% glycerol and heated in a boiling water bath (100^oC) for 2 min and analyzed by SDS-gel (8%) – electrophoresis as described in Chapter I.

The gels were stained with Coomassie blue-R and cut into 1 mm slices. The slices were digested with 0.5 ml of 30% hydrogen peroxide at 60^oC and radioactivity was measured using 0.5% PPO in Triton-toluene (1:2, v/v) mixture.

MATERIALS AND METHODS

Materials:

Phosphocellulose was purchased from Sigma Chemicals Company, U.S.A. All other chemicals used were of analytical grade.

Methods:

Preparation of soluble and membrane proteins from hypocotyls tissue :

IBA treated and control hypocotyls segments were used for the isolation of soluble and membrane fractions. The tissue (30-40 gms) was homogenized in ). O.O2M sodium phosphate buffer (pH 7.0) containing O.25M sucrose and 10 mM PMSF in a pre-cooled all glass-mortar and pestle. The homogenate was filtered through two layers of cheesecloth and the filtrate was centrifuged at 10,000 rpm for 10 min. The post-mitochondrial supernatant was again centrifuged at 42,000 rpm for 90 min. The supernatant (soluble fraction) was carefully removed. The membrane pellet was once rinsed with the homogenizing buffer and suspended in O.5 ml of the same.

Effect of cytochalasin B and colchicines on root initiation:

Hypocotyl segments (4-6 cm length) were treated with IBA (30 mG/ML) for 10 min. Then the segments were washed and placed in fresh nutrient medium. At different time intervals cytochalasin B (10 mg/ml) or colchicines (2.5 mg/ml) was added. After incubating for 6 hr, segments were removed and washed repeatedly in water and placed in fresh Hoagland’s medium. At the end of 72 hr, the roots formed were counted.

Isolation of tubulin from rat brain :

The isolation of tubulin from rat brain was carried out according to the methods of Weingarten et al. [244] With certain modifications. Adult rat brains were homogenized in 2 volume of PMS buffer (pH 6.8) (O.1M sodium pyrophosphate, O.O1M magnesium chloride and O.1M sucrose), and the homogenate was centrifuged at 10,000 rpm for 10 min. The supernatant was then spun at 42,000 rpm for 60 min in a preparative ultracentrifuge. The post-microsomal supernatant was carefully removed, mixed with an equal volume of Phosphocellulose (5 gm) equilibrated with PMS buffer (pH 6.8) and stirred slowly for 60 minutes. Then the mixture was centrifuged at 15,000 rpm for 15 min. The supernatant was removed and the Phosphocellulose pellet was washed successively with PMS buffer (pH 6.8) containing O.15M sodium chloride three times. The Phosphocellulose pellet was again washed three times with PMS buffer (pH 6.8) containing O.2M sodium chloride. Tubulin bound to Phosphocellulose was eluted with PMS buffer (pH 6.8) containing O.3-O.4M sodium chloride, by repeated centrifugation procedure. The eluate was dialyzed overnight against O.2M sucrose solution. The dialyzed tubulin was concentrated with sucrose and stored at –20^oC.

Isolation of tubulin from the hypocotyls segments of Phaseolus Vulgaris :

Plant tubulin was extracted by the same procedure as described for rat brain except that the PMS buffer used for homogenization and washing contained in addition 1 Mm DTT and 10mM phenylmethylsulphonyl fluoride. The post-mitochondrial supernatant was solubilized with the addition of deoxycholate to a final concentration of 1% and used for the isolation of tubulin. The Phosphocellulose used for adsorbing tubulin was also equilibrated with PMS buffer (pH 6.8) containing O.15M NaCl and 1% deoxycholate. After repeated washing (three times each) in PMS buffer containing O.15M NaCl and O.2M NaCl, tubulin was eluted with PMS buffer containing O.3M-O, 4M NaCl. The eluate was dialyzed overnight against O.2M sucrose, then the tubulin was concentrated with sucrose and stored at –20^oC.

Characterization of tubulin antibody:

Preparation of antibody to the rat brain tubulin :

Male albino rabbits (New Zealand strain) weighing about 2 Kg were injected with 2 mg of purified rat brain tubulin in Freund’s complete adjuvant. A booster dose was given after 21 days of the first injection. After seven days of the booster does, rabbits were bled from the marginal veins of the car. The serum was separated from the agglutinated blood by centrifugation at 10,000 rpm for 10 min and sodium azide (O.O2%) was added to the serum and stored at –20^oC.

Preparation of g - globulin :

The g - globulin fraction was prepared from the antiserum according to the methods of Campbell el al. (241). The antiserum was diluted with an equal volume of 0.2M sodium phosphate buffer (pH 7.5). Solid ammonium sulfate was added to 40% saturation and kept at –4^oC for 1 hr. Then, it was centrifuged at 10,000 rpm for 10 min. The precipitate was dissolved in 0.2M sodium phosphate buffer (pH 7.5) and dialyzed against the same buffer and passed through DEAE-cellulose column which was pre-equilibrated with the above buffer. The g - globulin fraction, that came out in the column volume was collected and reprecipitated by the addition of solid ammonium sulfate to 40% saturation. The precipitate was collected by centrifugation and dissolved in 0.02M Tris-HCl (pH 7.4) containing 0.15M sodium chloride and dialyzed against the same buffer. After adding sodium azide to a final concentration of 0.02%, the g - globulin fraction was stored in small aliquots at –20^oC.

Ouchterlony double diffusion analysis:

Ouchterlony plates were prepared according to Thomas el al. [242]. Agarose (0.9%) was dissolved in 1.0M glycine buffer (pH 7.4) containing 0.08M sodium chloride, 0.15M sodium Azide; heated to boiling and poured into Petri dishes (9 cm in diameter) to 3 mm thickness. Circular wells of required dimensions were made. The antibody preparation was put into the central well and the antigen in the peripheral, wells. The plates were left at room temperature for 1-2 hr and incubated at –4^oC for 48 hrs. After the precipitin lines had developed, the plates were washed repeatedly with 0.05M potassium phosphate buffer (pH 8.0) containing 0.3M sodium chloride and photographed.

Quantitative immunoprecipitation:

Membrane fraction from the hypocotyls segment was isolated as described earlier. The membrane fraction was solubilized in 0.02M Tris-HCl (pH 7.4) buffer containing 0.15M NaCl, 10 mM PMSF and 1% deoxycholate. Increasing concentrations of membrane proteins were dispensed into immunoprecipitation tubes and purified antibody (550 mg protein) raised against rat brain tubulin, was added to each tube. The final volume was made up to 0.5 ml with 0.02M Tris-HCl (pH 7.4) buffer. Then the tubes were incubated at 27^oC for one hour and at 4^oovernight. The immunoprecipitates were collected by centrifugation at 2500 rpm for 15 min. The immunoprecipitates were washed with 0.5 ml of 0.02M Tris-HCl (pH 7.4) buffer containing 0.15M NaCl. Separate blanks for membrane proteins and antibodies were also processed. The final immunoprecipitates were dissolved in 0.2 ml of 0.01N sodium hydroxide and protein was estimated according to the method of Lowry et al. [221].

Tubulin polymerization assay:

Tubulin polymerization was determined by GTPase assay, in presence of microtubule reassembly buffer [249]. The reassembly buffer in a total volume of 500 ml contained 5mM MES buffer (pH 6.8), 1mM EGTA, 0.5mM MgCl₂and 50 mm KCL, 100 mg of membrane protein and 20 mg of tubulin (rat brain or plant). After equilibrating the reaction mixture in a shaker water bath (270C) for 10 min, the reaction was initiated by the addition of GTP to 1 mm final concentration. At various time points the reaction was terminated by adding 50% TCA to a final concentration of 10%. Then the tubes were kept for 60 min at 00Cj. The precipitate was sedimented by centrifuging at 5,000 rpm for 10 min. The supernatant was carefully removed and used for estimating phosphate by the method of Fiske and Subba Roa [248]. Blanks for GTP, tubulin, and membrane fraction were also run along with the samples. For all combinations zero time controls were also maintained.

TUBULIN SYNTHESIS IN VIVO:

In order to quantify the synthesis of tubulin in response to IBA, hypocotyls segments (20 gm) were treated with IBA (30 mg/ml). After treating the segments with hormone for 30 min, the control and IBA treated segments were incubated 24 hr in 5 ml of nutrient medium containing 25 mCi of [¹⁴ C]-chlorella protein hydrolysate. Then the segments were thoroughly washed, homogenized and the membrane fraction isolated and immunoprecipitated as described. The immunoprecipitates were dissociated in equal volumes of SDS-buffer and analysed on SDS-gels (8%). The gels were then subjected to fluorography.

SDS-Gel electrophoresis:

Soluble proteins (250 mg each), membrane proteins (250 mg each) and immunoprecipitates were analysed in SDS-gel electrophoresis as described in experimental methods of Chapter I.

Fluorography of polyacrylamde gels:

After electrophoresis of the immunoprecipitates, the slab gels were impregnated with PPO, dried and fluorographed for 15 days at –20^oC using India X-ray films (40 ASA) basically according to the procedures described by Bonner and Laskey [246].

MATERIALS AND METHODS

Materials:

Phosphocellulose was purchased from Sigma Chemicals Company, U.S.A. All other chemicals used were of analytical grade.

Methods:

Preparation of soluble and membrane proteins from hypocotyls tissue :

IBA treated and control hypocotyls segments were used for the isolation of soluble and membrane fractions. The tissue (30-40 gms) was homogenized in). O.O2M sodium phosphate buffer (pH 7.0) containing O.25M sucrose and 10 mM PMSF in a pre cooled all glass-mortar and pestle. The homogenate was filtered through two layers of cheese cloth and the filtrate was centrifuged at 10,000 rpm for 10 min. The post-mitochondrial supernatant was again centrifuged at 42,000 rpm for 90 min. The supernatant (soluble fraction) was carefully removed. The membrane pellet was once rinsed with the homogenizing buffer and suspended in O.5 ml of the same.

Effect of cytochalasin B and colchicines on root initiation:

Hypocotyl segments (4-6 cm length) were treated with IBA (30 mG/ML) FOR 30 MIN. then the segments were washed and placed in fresh nutrient medium. At different time intervals cytochalasin B (10 mg/ml) or colchicines (2.5 mg/ml) was added. After incubating for 6 hr, segments were removed and washed repeatedly in water and placed in fresh Hoagland’s medium. At the end of 72 hr, the roots formed were counted.

Isolation of tubulin from rat brain :

The isolation of tubulin from rat brain was carried out according to the methods of Weingarten et al. [244] with certain modifications. Adult rat brains were homogenized in 2 volume of PMS buffer (pH 6.8) (O.1M sodium pyrophosphate, O.O1M magnesium chloride and O.1M sucrose), and the homogenate was centrifuged at 10,000 rpm for 10 min. The supernatant was then spun at 42,000 rpm for 60 min in a preparative ultracentrifuge. The post-microsomal supernatant was carefully removed, mixed with an equal volume of Phosphocellulose (5 gm) equilibrated with PMS buffer (pH 6.8) and stirred slowly for 60 minutes. Then the mixture was centrifuged at 15,000 rpm for 15 min. The supernatant was removed and the Phosphocellulose pellet was washed successively with PMS buffer (pH 6.8) containing O.15M sodium chloride three times. The Phosphocellulose pellet was again washed three times with PMS buffer (pH 6.8) containing O.2M sodium chloride. Tubulin bound to Phosphocellulose was eluted with PMS buffer (pH 6.8) containing O.3-O.4M sodium chloride, by repeated centrifugation procedure. The eluate was dialyzed overnight against O.2M sucrose solution. The dialyzed tubulin was concentrated with sucrose and stored at –20^oC.

Isolation of tubulin from the hypocotyls segments of Phaseolus Vulgaris :

Characterization of tubulin antibody:

Preparation of antibody to the rat brain tubulin :

Preparation of g - globulin :

Ouchterlony double diffusion analysis:

Ouchterlony plates were prepared according to Thomas el al. [242]. Agarose (0.9%) was dissolved in 1.0M glycine buffer (pH 7.4) containing 0.08M sodium chloride, 0.15M sodium Aizde; heated to boiling and poured into petri dishes (9 cm in diameter) to 3 mm thickness. Circular wells of required dimensions were made. The antibody preparation was put into the central well and the antigen in the peripheral, wells. The plates were left at room temperature for 1-2 hr and incubated at –4^oC for 48 hrs. After the precipitin lines had developed, the plates were washed repeatedly with 0.05M potassium phosphate buffer (pH 8.0) containing 0.3M sodium chloride and photographed.

Quantitative immunoprecipitation:

Membrane fraction from the hypocotyls segment was isolated as described earlier. The membrane fraction was solubilized in 0.02M Tris-HCl (pH 7.4) buffer containing 0.15M NaCl, 10 mM PMSF and 1% deoxycholate. Increasing concentrations of membrane proteins were dispensed into immunoprecipitation tubes and purified antibodies (550 mg protein) raised against rat brain tubulin, was added to each tube. The final volume was made up to 0.5 ml with 0.02M Tris-HCl (pH 7.4) buffer. Then the tubes were incubated at 27^oC for one hour and at 4^ooovernight. The immunoprecipitates were collected by centrifugation at 2500 rpm for 15 min. The immunoprecipitates were washed with 0.5 ml of 0.02M Tris-HCl (pH 7.4) buffer containing 0.15M NaCl. Separate blanks for membrane proteins and antibodies were also processed. The final immunoprecipitates were dissolved in 0.2 ml of 0.01N sodium hydroxide and protein was estimated according to the method of Lowry et al. [221].

Tubulin polymerization assay:

Tubulin polymerization was determined by GTPase assay, in presence of microtubule reassembly buffer [249]. The reassembly buffer in a total volume of 500 ml contained 5mM MES buffer (pH 6.8), 1mM EGTA, 0.5mM MgCl₂and 50 mm KCL, 100 mg of membrane protein and 20 mg of tubulin (rat brain or plant). After equilibrating the reaction mixture in a shaker water bath (270C) for 10 min, the reaction was initiated by the addition of GTP to 1 mm final concentration. At various time points the reaction was terminated by adding 50% TCA to a final concentration of 10%. Then the tubes were kept for 60 min at 00Cj. The precipitate was sedimented by centrifuging at 5,000 rpm for 10 min. The supernatant was carefully removed and used for estimating phosphate by the method of Fiske and Subba Row [248]. Blanks for GTP, tubulin, and membrane fraction were also run along with the samples. For all combinations zero time controls were also maintained.

TUBULIN SYNTHESIS IN VIVO:

SDS-Gel electrophoresis:

Soluble proteins (250 mg each), membrane proteins (250 mg each) and immunoprecipitates were analysed in SDS-gel electrophoresis as described in experimental methods of Chapter I.

Fluorography of polyacrylamde gels:

Experimental Procedures:

Materials: Electrophoretic grade Agarose and restriction enzymes ere purchased from Bethesda Research Laboratory. Ultra pure sucrose was from Schwarz/Mann. Sequanal grade NaDodSO4 and optical grade CsCl were purchase from Pierce chemicals Co. Poly (U- Sepharose 4B was purchased from Pharmacia. Methyl mercury hydroxide (1M solution) was from Alfa Ventron Corporation. Most of the inhibitors like Chloramphenicol, Ethidium bromide and Cordecepin triphosphate were purchased from sigma chemicals Co.[3H]CTP (18 Ci/mmol), and [32P] dCTP (>3000 Ci/mmol) were obtained Amersham Corp. Nitrocellulose membrane sheets for RNA blot transfer were purchased from Schleicher & Schuell.

Isolation of Mitochondria: Ehrlich ascites mouse tumor cells were grown in the peritoneal cavity of Swiss mice (Chun et al, 1969) and used as the source of mitochondria. Disruption of cells by homogenization in sucrose-mannitol buffer (4mM HEPES, pH 7.4, 220mM mannitol, 70mM sucrose, and 2mM EDTA) and isolation of crude mitochondria by differential centrifugation were as described before (Lewis et al, 1976, Niranjan and Avadhani, 1980; Bhat et al, 1981). Mitochondria were washed once with sucrose-mannitol buffer containing 20mM EDTA, and the mitoplasts were isolated by the digitonin (fractionation method using 0.1 mg of digitonin/mg of mt. protein, also as described before (Bhat et al, 1982).

In vitro labeling of mitoplasts and isolation of mt RNA: mt RNA was labeled with 3H-labeled or32P-labeled nucleotides by using an in vitro system previously described for labeling mt translation products (Bhat et al, 1981, 1982). Freshly prepared mitoplasts were suspended in the RNA synthesis buffer [5mMhepes,pH7.4, 60mM KCl, 6mM Mg (CH3-COO) 2, 5 mM 2-mercaptoethanol, 3 mM KH2PO4, pH 7.4 and 0.14 M sucrose] at a concentration of 6-10 mg of mitoplast protein/ml and supplemented with 2mM ATP, 1 mM GT, 5mM creatine phosphate, 4 mM pyruvate, 0.2 mg/ml creatine phosphokinase, and 100 uM each of 20 amino acids. The mixture was gently shaken at 35^oC, and the labeling was initiated by adding 100 uCi/ml each of [3H] CTP (18 Ci/mmol), and [3H] UTP (50uCi/mmol) or 100 uCi/ml [32p] UTP (>600 Ci/mmol). Unless otherwise stated, labeling was continued for 60 minutes. Aliquots (2.5 uL) were withdrawn at intervals, adsorbed onto filter discs, and assayed for radioactive RNA synthesis by cold Ccl3COOH method (Mans & Novell, 1961).

The labeled mitoplasts were pelleted at 10 000g for 10 min at 4oC, washed once with sucrose-mannitol buffer, and used for isolation of mt RNA by phenol-chloroform method (Avadhani, 1979) with the following modifications. The mt pellet (5-10mg) was suspended in 2.5ml of guanidinium thiocyanate buffer (25mM sodium citrate, pH7.0, 5M guanidinium thiocyanate, 0.1M 2-mecrcaptoethanol, and 0.5% sodium laurylsarcocinate) by homogenization with Dounce homogenizer (Chirgwin et al, 1979). The clear lysate was extracted with equal volumes of phenol saturated with water and chloroform. The aqueous phase was separated and saved, and the interphase was resuspended in guanidinium thiocyanate buffers above and extracted again with phenol-chloroform. The combined aqueous phases were rextracted once with phenol-chloroform and once with CHCl3-isoamyl alcohol (95:5) and adjusted to pH 5.0 by adding 0.025 volume of1 MCH3COOH. RNA was precipitated with 2 volumes of ethanol in the presence of 0.3M CH3COOK (pH 5.0). RNA was further pelleted through CsCl (Chirgwin et al, 1979) to eliminate contaminating DNA.

Preparation of Plasmid DNA: E.coli C600r-m- transformed with pACYC 177 plasmids carrying the entire mouse mt genome (designated as pAM1) was a gift from Dr. David A.Clayton (Martens & Clayton, 1979). The growth of cells in PO4 L broth and lyses of cells with Lysozyme-EDT and triton X-100 were according to Battey & Clayton (1978). The lysate was clarified at 75000g for 30 min at 4oC and was made to 10% with polyethylene glyco). The nucleic acid precipitate was collected by centrifugation at 25000g for 30 min at 4oC, digested with pancreatic RNase (DNase free), and used for the isolation of closed DNA by CsCl banding (Clewell & Helinski, 1970).

Isolation of Nick Translation of Restriction Fragments: The plasmid DNA containing mouse mt genome (10-50ug) was digested to completion with EcoR1 and Bgl I under standard conditions recommended by the vendor. The DNA fragments were resolved by electrophoresis on 0.6% Agarose slabs and localized by staining with 0.5ug/ml EtBr. The DNA from the gel slices was electro blotted on DEAE paper (NA 45, Schleicher & Schuell), eluted by extraction with 1M NaCl, and precipitated with 2.5 vol of ethanol. The DNA fragments were Nick translated with [32P]-dCTP (>3000Ci/mmol) by using a kit supplied by Bethesda research laboratory.

Covalent Binding of DNA to Cellulose: About 200-250 g of mt DNA (restricted fragments) in 1ml of H2O was sonicated for 10s and denatured by heating at 100oC, followed by quick chilling in ice. The single stranded DNA fragments were covalently linked to epoxy-cellulose using the method of Moss et al,(1981).

Electrophoretic Analysis of RNA: RNA was separated on denaturing Agarose methyl-mercury hydroxide gels (Baily & Davidson, 1976).

After the electrophoresis run, the gels were stained with 1ug/ml ethidium bromide in 0.5m NH4CH3Coo, and the RNA bands were visualized under UV light. 3H-labeled RNA bands localized by fluorography by using EN3HANCE (New England Nuclear). In northern blotted experiments (Alwine et al, 1977), 1977, NA from the gels was blotted onto nitrocellulose sheets as described by Thomas (1980) and probed with 32P-labeled DNA fragments (Alwine et al, 1977).

DNA-RNA hybridization: DNA (2-10ug) in 100ul of 0.3M NaCl and 0.3M NaOH was denatured by heating at 100oC for 15 min following quick chilling in ice. DNA was spotted on nitrocellulose disks and immobilized by heating at 80oC under vacuum for 2h. Hybridization was carried out in 0.5-1 ml of reaction mixture containing 5 x SSPE9 (1XSSPE=0.18M NaCl, 10mM sodium phosphate, pH 7.1, and 1mM EDTA), 50% formamide, 2 2x Denhardt’s solution [0.04% each of Ficoll, poly (venylpyrrolidone), and bovine serum albumin], 50ug/ml yeast RNA at 42oC for 48hr. The filters were washed with 25ml of 2x SSPE and 25ml of 1x SSPE. The filters were digested with 50 ug of pancreatic RNase in 1ml of 1x SSPE, washed with 10ml of 1x SSPE, air dried, and counted with 10ml of ACS II scintillation mixture Amersham).

EXPERIMENTAL

Morphological studies. Phaseolus vulgaris L. var. Butpees atcingtesa stingless raised in sand in a dark chamber. When the hypocotyls attained a height of 20-25 cm, they were cut l5 cm below the cotyledons and washed for 30 min in running H₂O. Hypocotyl segments of 4 or 8 cm length were kept in a suitable vol. of half strength Hoagland's nutrient soln. (8) with or without IBA. In all the long term experiments; a 12 hr day period was maintained . The medium was changed every day: To reduce bacterial contamination, 30 g/ml of Ampicillin (Hindustan Antibiotic Limited) was used. The number of root initiate wart counted at different periods of time.

For anatomical studies serial hand sections were made, stained with 2 a (W/V) sacrament and observations were made under the microscope.

Protein synthesis in vivo: Protein synthesis in the hypocotyls tissue was followed by placing 1 g of suitably treated and washed segment in 5 ml medium containing 10Ci of (¹⁴C)-chlorella hydrolysate. The different treatments given are described in the table. After 30 min of labeling, the tissues were thoroughly washed to remove adhering radioactivity and a tissue homogenate was prepared by grinding in NaPi buffer (0.1 M pH 7) in the cold. HCHO4 (0.3 N in final conc.) was added to the homogenate and the ppt, was recovered by centrifugation after standing a few hr in the cold. Protein was extracted from the ppt. with NaOH; the extract neutralized and re-precipitated with TCA (10%w/v, in final concentration). The precipitate was subsequently washed with hot TCA, EtOH-Et2O (3:1) and finally with Et2O. Final preparation was dissolved in HCO2H and aliquots were counted on whatman No 3 filter paper discs. Aliquots were also used after neutralization for measurement of protein content by the method of ref [9]. The specific radioactivity of the amino acid pool was estimated in the HclO4 supernatant. The supernatant was neutralized and aliquots were used for radio activity measurement and total amino acid was estimated using ninhydrin by the procedure of ref.[10].

Protein synthesis in vivo: For this purpose polysomal fraction was isolated from the hypocotyls tissue. Isolation procedures and in vitro amino acid incorporation were similar to those described if ref.[11]. Briefly, the tissue was homogenized in 5 vols. Of buffer A containing Tris-HCL, pH 7.5, 50mM; MgCl2, 5mM; KCl, 15mM; DTT, 0.6mM and sucrose, 300mM. The homogenate was centrifuged at 20,000g for 15 min and 2ml of the supernatant was layered over 3 ml of 1.5M sucrose and centrifuged at 150 000 g for 2hrs. The pellet was suspended in buffer A. Another batch of homogenate was centrifuged at 20 000g for 15 min and the supernatant centrifuged at 150 000g for 2 hrs. The final supernatant was passed through a G-25 column and was used as the source of soluble enzymes (S150 fraction). The in vitro protein synthesizing incubation mixture contained in 0.1 ml total vol: HEPES buffer, pH 7.5, 20mM; MgCl2, 5mM; KCl, 50mM; ATP, 0.4mM; GTP, 0.12mM; phosphoenolpyruvate, 5mM; pyruvate kinase, 2 enzyme unit; amino acid mixture without Leucine, 25 uM each amino acid, polyribosome, 100ug, RNA; S150 fraction, 100 ug protein; 1uCi of Leucine [3H](7Ci/mMol). The mixture was incubated at 37^C for 30 min.

Aliquots (20ul) were applied to filter paper discs and discs after drying were successively washed with hot and cold 5%TCA, EtOH-Et2O 92:1) and finally Et2O. The discs were counted for radioactivity.

RNA synthesis: It was measured using either carrier free 32P-orthophosphate (200uCi/10 ml/10g tissue) or Uridine [3H] (500 uci/5ml/5g tissue). The labeling period was 30 min. After thorough washing, RNA was isolated from the tissue homogenate using PhOH-CHCl, extraction procedures [12]. Poly (A)-containing RNA was isolated from a known amount of total RNA using poly (U)-Sepharose columns [13]. For measurement of nucleotide pool sp.radioactivity in experiments using 32P, the tissue homogenate was treated with HClO4 (0.3N final conc.). After standing for a few hr in the cold, it was centrifuged and the free nucleotides in the supernatant was adsorbed using activated charcoal.. The charcoal pellet was washed repeatedly with H2O and finally the nucleotides were eluted using 50% EtOH, 0.3N NH4 OH [14]. Aliquots of the eluate were used for radioactivity and A260 measurements. Radioactivity of the filter paper discs was measured in a scintillation counter with 10 ml of toluene containing 0.5% PPO (w/v) with an efficiency of 6% for 3H and 35% for 14C.

EXPERIMENTAL

Seeds of Phaseolus vulgaris L. were purchased from the Lalbagh Seed Nursery. Bangalore. India. ¹⁴C-Labelled Chlorella protein hydrolysate (27 Ci/m atom C) was purchased from Bhaba Atomic Research Centre; Bombay [³H] Leucine (105 Ci/mmol) was purchase from the Radiochemical Centre,

Amersham. U.K. Oligo dT-cellulose was purchased from Collaborative Research Inc., Waltham, and U.S.A. All other biochemicals were purchased from Sigma.

Effect of colchicine, cytochalasin B and actinomycin D on root production. The conditions for root production in the hypocotyls segments of Phaseolus vulgaris have been described in ref. (8). Briefly, the hypocotyl segments (8-10 cm) obtained from the seedlings were washed thoroughly and then placed in half strength Hoagland's nutrient tedious. Hormone treatment involved the addition of IBA to the medium at a conc. of 10 g/ml and the hypocotyls were exposed for 30 min. They were then washed thoroughly, placed in fresh medium without the hormone. And the number emerging roots was counted after 72 hr. To prevent bacterial contamination. Ampicillin (30g/ml) was added. The effects of Actinomycin D (l0g(ml), cytochalasin B (10ug/ml, and colchicines (2.5mg/ml) on root production were tested by adding these components to IBA pretreated hypocotyls after different periods. The hypocotyls were exposed to these compounds for 6hrs, washed thoroughly, and then transferred to fresh media without the inhibitors. The number of emerging roots in each case was counted at the end of 72 hrs.

Quantitation of translatable messenger activity: For this purpose, total and poly(A-containing RNAs were isolated from control and IBA pretreated hypocotyl segments using phenol-CHCl3 extraction procedure [12] Poly(A)-containing RNA was isolated from total RNA as described in ref[13].

The messenger activity of the RNA preparation was assayed in the wheat germ cell-free system [14]. The assay mixture in a vol. of 100 ul contained Hepes, 20 M; GTP, 20 M; ATP 1 mM; Phospho creatine, 8 mM; Mg (OAc)₂, 3 mM; KCI, 100 mM; DTT, 2 mM; amino acid mixture without Leucine, 25 M each; (105 Ci/mmol), 20 uCi; and S-30 fraction, 1 A:60 unit of RNA (an optimum concn. in the range giving a linear response). The mixture was incubated at 25° for 60 min and 5 ul portions were transferred to Whatman filter paper discs. Washed with hot and cold CCI₃COOH (TCA) and then with Et₂O twice, and used for measuring radioactivity incorporated into total proteins. The radioactivity incorporated into the TCA perceptible protein product at identical concentrations of different RNA preparations is taken as the measure of messenger activity.

Radioactivity profile of the cell free products: The labeled cell free products were precipitated with cold TCA (10 % w/v in 6na1 concn) and washed with Et₂O. The pellet was dissipated with 2 % (w/v) SDS containing 10 mM Tris-HCI (pH 6.8), 5 %  mercaptoethanol and 10% (v/v) glycerol at 100° for 2 min. The protein products were analyzed using 8 % SDS-polyacrylamide slab gels [15].The gels were stained with Coomassie blue, distained, and then each slot was cut into 1.5 mm slices. The slices were digested with 0.5 ml H₂O₂ (30% soln) at 60° overnight and the radioactivity was measured using 0.5 % (w/v) 2,5-diphenyloxazole in Triton-toluene (1:2, v/v) as solvent.

Analysis of membrane proteins: For this purpose, IBA pretreated and control hypocotyl segments were homogenized in 0.25 M sucrose containing 20 mM KPi buffer, pH 7.2 and 1 mM phenylmethylsulphonyl fluoride. The homogenate was filtered through 2 layers of cheesecloth and the filtrate was centrifuged at 10 000 g for 15 min. The supernatant was spun at 105 000 g for 90 min and the pellet was used as the microsomal fraction. The proteins were analyzed using 8% SDS-polyacrylamide slab gels.

Tubulin polymerization: It was followed by assaying for two parameters. In one case, the attendant GTPase activity was measured in the presence of microtubule reassembly buffer [9]. The reassembly buffer in a total vol. of 500 l contained 5 mM morpholine ethane sulphonate (MES) buffer (pH 6.8), 1mM, EGTA. 0.5 mM MgCI₂ 50 mM KCI, 100 g of microsomal protein fraction and 20 g of tubulin obtained from either rat brain or hypocotyl segments. After the reaction mixture was equilibrated in a shaking water bath at 27% for 10 min, the reaction was initiated by the addition of GTP (1 mM in final concn). The reaction was terminated at different time intervals by adding 50% TCA to a final concn of 10%. The tubes were kept at 0⁰C for 30 min, centrifuged, and the supernatant was used for the estimation of Pi content by the method of ref. [16].

In the other method, tubulin polymerization was followed using the turbidometric assay procedure [17]. Phosphocellulose purified plant tubulin (1 mg/ml) was incubated in MES buffer (100 mM MES, 0.5 mM MeC1₂=, 1 mM EGTA and 50 mM KCI, pH 6.8) in the presence of microsomal membranes from control and IBA-pretreated hypocotyls at 37°. The reaction was initiated by the addition of GTP and A was measured at 350 mm as a function of time.

Synthesis of tubulin in vivo: After 18 hr of incubation in half strength Hoagland's medium, both control and IBA-pretreated hypocotyl segments (2-4 cm length) were placed in 5 ml of fresh medium containing 25 Ci of ¹⁴C-Iabeled Chlorella protein hydrolysate. After another 6 hr, the segments were thoroughly washed and the microsomal fraction was isolated. The membrane proteins were rendered soluble in 20 mM Tris-HCI (pH 7.4) containing 0.15 M NaCl, 1% Na deoxycholate and 1 % Triton X-100. Immunoprecipitation of tubulin was carried out by the addition of a 2-fold excess of anti-tubulin I₈G based on the equivalence point. The incubation was carried out at 37° for 30 min and then at 4° overnight. The ppts were washed with the immunoprecipitation buffer and finally with Tris-NaCl buffer. The immunoprecipitate was then analyzed on SDS-polyacrylamide gels (8%) as described earlier and subjected to fluorography [18]. The tubulin bands were also sliced, digested with H₂O₂ and the radioactivity was measured.

Other procedures:

Antibodies were raised in rabbits for rat brain tubulin prepared according to the method of ref. [19], with certain minor modifications. The IgG fraction was prepared from the antisera and its cross-reaction with purified plant tubulin and the plant microsomal membrane fraction was ascertained by the Ouchterlony technique. Plant tubulin was isolated from IBA pretreated hypocotyl segments by the procedure employed for the isolation of rat brain tubulin. An SDS-polyacrylamide slab gel Electrophoretic profile of the final preparation is given in Fig.5. The anti-tubulin IgG was titrated against the plant tubulin. I8G was also titrated against the plant microsomal membrane proteins rendered soluble by procedures described above and at the equivalent point, 500 g 1gG protein was found to immunoprecipitate 3.2 mg of the microsomal proteins quantitatively. Protein content was measured by the method of ref. [20].

METHODS

Materials: French bean seeds were purchased from a local seed nursery. Hypocotyl segments of 3 - 4 cm length from the dark grown seedlings were cut. Then they were abated of auxin by washing in distilled water for 30 mins and made certain that none of the segments produced any new roots on their own. Such segments were incubated for 30 mins in half strength Hoagland's medium (Arnold, 1968) containing 5 x 10^-5 M IBA or 4·5 x 10^-6 M BAP or both. Then the segments were surface washed and further incubated in nutrient medium for the required durations. Aseptic condition was maintained strictly in all experiments.

¹⁴C -labeled Chlorella hydrolysate (270/m atom C) and ³²p - carrier free orthophosphate were purchased from Bhaba Atomic research Centre, Bombay. ³H –Leucine (105 Ci/mM was) purchased from Radiochemical Centre, Amersham., U.K., Oligo dT-cellulose and Poly(U)-Sepharose were purchased from Collaborative research Inc., Waltham, U.S.A All other biochemicals were purchased from Sigma, U.S.A

Rate of protein synthesis and RNA synthesis: Hormone pre-treated and control segments at different stages of development were exposed to ¹⁴C Chlorella hydrolysate or 3H - UTP or ³²P - orthophosphate for 30 mins; then they were surface washed and processed for determining the protein synthesis by the methods of Rosen, 1957 and Lowery, 1957, and RNA synthesis by the methods of Pennman, 1966 and Padmanabhan et al., 1975·

Quantification of mRNA activity and in vitro protein: synthesis Isolated total RNA or poly (A) ⁺RNA were translated in a cell free system derived from wheat germ using ³H - Leucine as the label by Roberts and Patterson method, 1973.

Protein analysis: Membrane proteins and in vitro radiolabel led poly (A)+RNA translated proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by the methods of Laemli, 1970. For measuring radioactivity of proteins in the gels, the gels were first stained with Coomassie brilliant blue and then the required gels were sliced to 1-mm pieces, and digested, and counted for radioactivity.

METHODS

¹⁴C -labeled Chlorella hydrolysate (270/m atom C) and ³²p - carrier free orthophosphate were purchased from Bhaba Atomic research Centre, Bombay. ³H –Leucine (105 Ci/mM was) purchased from Radiochemical Centre, Amersham., U.K., Oligo dT-cellulose and Poly(U)-Sepharose were purchased from Colloborative research Inc., Waltham, U.S.A All other biochemicals were purchased from Sigma, U.S.A

Protocols for Generating Transgenic Plants

Isolation and Purification of Plant Viruses:

Tomato plants infected with Leaf curl virus (Gemini viruses) and Tomato mosaic virus (ToMV) were collected from the field from various locations in Karnataka and Delhi. Leaves from severely infected plants were sorted out. Isolation and purification of these viruses is done separately.

Leaves were thoroughly washed in tap water then with distilled water and frozen. The frozen leaves were homogenized in chilled mortar and pestle in 100 mM Tris-Cl buffer, pH 7.4 with 100mM NaCl, and 50mM EDTA and the slurry was filtered through two-layered muslin cloth. The filtrate was centrifuged at 15000 rpm for 15 min. The supernatant was aspirated and made with polyethylene glycol 7000 to 5x and gently stirred for about 3hrs. The slurry was centrifuged at 15000 rpm for 15 min. The pellet was then suspended n Tris-Cl-NaCl buffer.

The suspension obtained from Gemini viral and ToMV infected leaves were layered on step gradient (20% to 60%) Sucrose buffered with Tris-Cl – NaCl pH 7.2. and centrifuged at 42000 rpm in a swing out rotor for 3 hrs in Beckman ultracentrifuge. Then each layer of the step gradient sucrose solution with a syringe was carefully. The same was diluted and precipitated with PEG 7000, and each fraction was pelleted. The pellets were resuspended in Tris-buffer. Few micro liters of each sample were taken out for electron microscopic observation. Each fraction is suspended in sample solution for SDS PAGE, boiled for 2 min and dispensed into wells of the gradient (6-14%) SDS-polyacrylamide gel and electrophoresed. The same was fixed in 25 TCA and stained with Coomassie blue and destained with 80% EtOH and 20% ether. From the stained gel the identity of the viral protein is determined by their mol weight. Gemini virus showed 32Kd and ToMV showed 38KD. The fractions containing only viral particles were pooled and used for isolation nucleic acid; DNA from the Gemini virus and RNA from ToMV by known Tris-Cl-SDS and Phenol-chloroform methods. The nucleic acids were run in a 0.7% Agarose gel containing EtBr and Nucleic acid ands were viewed. Electron microscopic examination showed the presence of Gemini virus and ToMV in 30% and 40% sucrose fractions, which was confirmed by their capsid protein size and nucleic acid fraction.

Cloning of Gemini and ToMV capsid and non functional capsid genes:

Using specific primers single stranded Gemini DNA was made into double stranded structures, similarly RNA was reverse transcribed to generate double stranded cDNA. The same were restricted digested to isolate capsid protein coding segment (used the available DNA and RNA sequence of Gemini and ToMV particles). The DNA segments were cloned into Bgl II site in the PGA472 plasmid, next to a strong CAMV 35s promoter, (a gift from Veluthambi, Professor, Dept. of Plant Biotech, Madurai Kaamaraj University, Madurai). The cloned plasmid was used for transformation of E.coli bacteria and confirmed the presence of the plasmid by isolating the pGA472 plasmid. PGA 472 is a binary vector containing sites for inserting any given DNA next to 35S CAMV promoter elements and also it had Hygromycin resistance selection marker gene under the control NoS promoter elements. This Protocol is to generate capsid protein mediated resistance In another modification the cloned capsid genes were trimmed from 5’ end of the gene and also from 3’ end, and cloned into to the same sites in the binary vectors. They are found to generate non-functional RNA. This is to generate non-capsid protein mediated resistance or RNA mediated resistance. Today we know the RNA mediate resistance id due to micro RNA or RNAi mediated resistance. The same plasmids were used to transform Agrobacterium LB 4404 by triparental mating protocol. The presence of the Plasmid was again confirmed by the isolation of the plasmid from the Agrobacterium.

Tomato plants were tissue cultured using sterile seedlings. The callus was used to transfect the plasmid with Agrobacterium. After co-culturing the bacteria with the callus, the same was washed in sterile distilled water and cultured in MS medium containing carbamycin, and Hygromycin for two days and the callus is repeatedly transferred to fresh medium till no adhering Agrobacteria were found. The callus was regenerated in MS media containing 0.5mg/L IAA and 2mg/L BAP (Benzene Amino Purine). The shots regenerated in the presence of Hygromycin, were harvested and DNA was extracted. The same was used PR amplification using primers obtained for Gemini viral DNA and ToMV RNA. Furthermore whether or not the transgenic plants expressed viral proteins, western blot was performed using specific antibodies to the respective capsid proteins. This was also confirmed the transgenic nature of the plants. The regenerated plants were thus confirmed as transgenic plants. After Micropropogation the transgenic plants, they were hardened in green house and transferred into field conditions, where they grew well along with other non-transgenic plants with infections. For a long time and repeated growing the Tomato-TG plants, they are found to be resistant to the virus. Later the plants resistant to Gemini virus and Plants resistant to ToMV viruses were crossed and obtained double resistant tomato-T G hybrids. However the inserts were found to be unstable after three to four generations, so fresh transgenic plants have to be obtained de novo.

Cloning and Sequencing of HOX genes:

The primers of all the eight Hox genes were used for cloning them form Fishes. DNA was extracted from different species of Fish tissues. About 2ug of DNA from each sample was used for amplification, the amplified DNA was eluted by gel extraction kit provided by Promega. The size of each PCR product was evaluated by 0.8% Agarose gels and documented. The same DNA is legated to plasmids compatible for the PCR product for the Vectors have legating sequences with the 3’ A extension in PCR DNA. The ligated DNAs were purified and E.coli plasmids JM 109 were used for transformation using competent cells from Promega. Clones obtained were grown in culture and Plasmids were extracted by alkaline lyses method and the same were analyzed on Agarose gels.

The purified Plasmid DNA was used for sequencing. About 50-70 ng of DNA was used for sequencing. Promega sequencing kit with Big-dye was used for PCR sequencing, the reaction was initiated by Hot-start method and 32 cycles were performed. The reaction mix was loaded on to sequencing gels and read by automated sequencer and the sequences were recorded and compared for deriving Phylogenetic Tree among different specie of Fishes in the vicinity of Tulsa and nearby areas in Oklahoma State. Nearly 1500 sequencing reaction was conducted.

Protocols from websites for the use of Students:

Preparation of Genomic DNA from Bacteria- using Phase Lock Gel^TM

^{(Modified from Experimental Techniques in Bacterial
Genetics, Jones and Bartlet, 1990)}

Materials: see Solutions for Recipes

	TE buffer
	10% (w/v) sodium dodecyl sulfate (SDS)
	20 mg/ml proteinase K
	phenol\chloroform (50:50)
	isopropanol
	70% ethanol
	3M sodium acetate pH 5.2
	Phase Lock Gel^TM (Eppendorf-Brinkmann) (http://www.eppendorf.com/phaselockgel/en/phase_1.html)
	Grow E. coli culture overnight in rich broth. Transfer 2 ml to a 2-ml micro centrifuge tube and spin 2 min. Decant the supernatant. Drain well onto a Kimwipe. Resuspend the pellet in 467 μl TE buffer by repeated pipetting. Add 30 μl of 10% SDS and 3 μl of 20 mg/ml proteinase K, mix , and incubate 1 hr at 37 ° C. Add an equal volume of phenol/chloroform and mix well but very gently to avoid shearing the DNA by inverting the tube until the phases are completely mixed. CAUTION: PHENOL CAUSES SEVERE BURNS, WEAR GLOVES GOGGLES, AND LAB COAT AND KEEP TUBES CAPPED TIGHTLY. Carefully transfer the DNA/phenol mixture into a Phase Lock Gel^TM tube (green) and spin at 12,000 RPM for 10 min. Transfer the upper aqueous phase to a new tube and add an equal volume of phenol/chloroform. Again mix well and transfer to a new Phase Lock GelTM tube and spin 10 min. Transfer the upper aqueous phase to a new tube. Add 1/10 volume of sodium acetate. Mix. Add 0.6 volumes of isopropanol and mix gently until the DNA precipitates. Spool DNA onto a glass rod (or Pasteur pipet with a heat-sealed end). Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec. Resuspend DNA in at least 200 μl TE buffer. Complete resuspension may take several days. Store DNA at 4 ° C short term, -20 or -80 ° C long term. After DNA has dissolved, determing the concentration by measuring the absorbance at 260 nm.

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PCR Amplification of DNA

Materials:

sterile water

10X amplification buffer with 15mM MgCl2

10 mM dNTP

50 μM oligonucleotide primer 1

50 μM oligonucleotide primer 2

5 unit/μl Taq Polymerase

template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl

mineral oil (for thermocyclers without a heated lid

1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube:

10X PCR buffer	10 μl
Primer 1	1 μl
Primer 2	1 μl
dNTP	2 μl
template DNA and water	85.5 μl
Taq Polymerase	0.5 μl

2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water.

3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction.

4. Place tubes in a thermal cycler preheated to 94 degrees C.

5. Run the following program:

94 degrees C 1 min

55 degrees C 1 min or annealing temperature appropriate for particular primer pair

72 degrees C 1 min (if product is <500 bp), 3 min (if product is >500 bp)

for 30 cycles.

Program a final extension at 72 degrees C for 7 min.

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Phenol/chloroform Extraction of DNA

Materials:

	phenol:chloroform (1:1)
	chloroform

Add an equal volume of buffer-saturated phenol:chloroform (1:1) to the DNA solution.
Mix well. Most DNA solutions can be vortexed for 10 sec except for high molecular weight DNA which should be gently rocked. (If using Phase-Lock Gel, follow procedure M.1)
Spin in a microfuge for 3 min.
Carefully remove the aqueous layer to a new tube, being careful to avoid the interface. (Steps 1-4 can be repeated until an interface is no longer visible).
To remove traces of phenol, add an equal volume of chloroform to the aqueous layer.
Spin in a microfuge for 3 min.
Remove aqueous layer to new tube.
Ethanol precipitate the DNA( see ethanol precipitation)

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Procedure for Transfection of Mammalian Cells

Materials:

	Lipofectamine (Invitrogen)
	IMDM containing 10% fetal bovine serum, 1% glutamine, 1% aa
	IMDM containing 1% glutamine
	IMDM containing 20% fetal bovine serum, 1% glutamine, 1% aa

In a six-well or 35 mm tissue culture plate, seed ~2x 10⁵cells per well in 2 ml IMDM containing 10% FBS and nonessential amino acids.
Incubate the cells at 37°C in a CO₂ incubator until the cells are 70-80% confluent. This will usually take 18-24 h.
Prepare the following solutions in 12 x 75 mm sterile tubes:

Solution A: For each transfection, dilute 2 μg DNA (plasmid) in 375 μl serum-free IMDM (containing nonessential amino acids).
Solution B: For each transfection, dilute 12 μl LIPOFECTAMINE Reagent in 375 μl serum-free IMDM.

Combine the two solutions, mix gently, and incubate at room temperature for 15-45 min. The solution may appear cloudy, however this will not impede the transfection.Wash the cells once with 2 ml serum-free IMDM.
For each transfection, add 750 μl serum-free IMDM to each tube containing the lipid-DNA complexes. Do not add antibacterial agents to media during transfection. Mix gently and overlay the diluted complex solution onto the washed cells.
Incubate the cells for 5 h at 37°C in a CO₂ incubator.
Add 1.5 ml IMDM with 20% FBS without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium.Replace medium at 18-24 h following start of transfection.
Assay cell extracts for gene activity 24-72 h after the start of transfection, depending on cell type and promoter activity.

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Recommended Cycle Sequencing Protocols For ABI 3100

Template Quantity

Template	Quantity
PCR product: 100-200bp 200-500bp 500-1000bp 1000-2000bp >2000bp	1-3 ng 3-10 ng 5-20 ng 10-40 ng 20-50 ng
Single-stranded	25-50 ng
Double-stranded	150-300 ng
Cosmid, BAC	0.5-1.0 mg
Bacterial genomic DNA	2-3 mg

Reaction Mixtures
Reagent	Full Reaction	Half Reaction	Half Reaction(10 μl)
Big Dye Premix	8 μl	4 μl	2 μl
Big Dye Seq. Buffer	-	2 μl	1 μl
Template	See Table above	See Table above	See Table above
Primer (10 μM)	1 μl	1 μl	1 μl
Water	q.s.	q.s.	q.s.
Total	20 μl	20 μl	10 μl

Primer Quantity

Primer needed = 3.2 - 10 pmoles

PCR Cycle Sequencing Settings for Big Dye V3.1

Initial denaturing	96°C	1min
25 cycles of	96°C	10sec
	50°C	5sec
	60°C	4min
Hold at	4°C

Ethanol/EDTA Precipitation to clean up reactions

Add 5uL of 125 mM EDTA. Make sure the EDTA reaches the bottom of the tube.
Add 60uL of 100% ethanol to each tube.
Finger vortex and incubate at room temperature for 15 min.
Spin samples in a microcentrifuge at max speed in 4 ° C for 20min.
Carefully aspirate off the supernatant.
Add 60 μl of 70% Ethanol.
Spin samples in a microcentrifuge at maximum speed at 4 ° C for 15 minutes.
Aspirate off the supernatant.
Dry sample for 15 minutes in a Speed-Vac (longer if air-drying). Protect samples from light while they are drying.

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Colony PCR

Prepare a master mix containing the following:

1X reaction:

2.5 µl 2 mM dNTPs
2.5 µl 10 µM primer 1
10 µl 2.5X cresol red loading dye
2.5 µl 10X PCR buffer
5.0 µl sterile water
0.5-1 µl Taq polymerase

Note: If using PuReTaq Ready-To-Go PCR Beads (Amersham Biosciences), use 10 µl of water instead of dNTP’s, buffer and enzyme]

To prepare the master mix, multiply the volumes above by the number of colonies to be screened + 1.

Aliquot to 0.2 ml labeled PCR tubes and keep on ice.
Prepare one selection plate, e.g., LB + ampicillin, for every 20 colonies screened.
Label the plate with a grid so that each colony can be associated with a number that matches the number on the PCR tube and can be retrieved once PCR results are known.
With a toothpick or sterile loop, pick colonies from transformation plate, patch onto the selection plate and then place remainder in PCR with the same identifier.
PCR cycle using the same conditions for the original PCR with those same primers with one modification: include a 5 min 94°C denaturation at the beginning of the cycling reaction.
Analyze PCR results by running reactions directly onto an agarose gel (no additional loading dye is required)

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One-Step Gene Assembly

(Reference: Gang Wu, Julie B. Wolf, Ameer F. Ibrahim, Stephanie Vadasz, Muditha Gunasinghe and Stephen J. Freeland, Simplified gene synthesis: A one-step approach to PCR-based gene construction, Journal of Biotechnology, 124(3):496-503)

Design overlapping oligonucleotides, generally 40 bases in length, that encompass the sense strand of the gene of interest. Design antisense oligonucleotides that stagger the sense oligos by 20 bases.
Design outside PCR amplification primers to incorporate the appropriate restriction enzyme recognition sites, if desired, and to overlap the assembled gene sequence by at least 15 nucleotides. (Order oligonucleotides from IDT or Invitrogen using their standard desalting purification).
Reconstitute oligonucleotides to 100 µM in 10 mM Tris pH=8.5 (same as Qiagen buffer EB). Vortex well to reconstitute and store at -20°C.
Prepare a gene assembly mix by combining 5 µl of each of the gene assembly oligos. Dilute this mix so that each oligo is at a final concentration of 1 µM. (This will be referred to as 1X). Then dilute this mix 1:2 (0.5X), 1:5 (0.2X) and 1:10 (0.1X) in Tris buffer. This step is to optimize the assembly/amplification of the required product which varies from one gene to the next – so best to set up 4 different reactions, one for each dilution of the mix, so determine which gives you the best yield and the least background.
Prepare 10 µM dilutions of outside amplification primers.
Perform one step gene assembly/amplifications using the following:

Reaction conditions:

Template	5 µl gene assembly mix (1X, 0.5X, 0.2X, and 0.1X)
Primers (0.4 uM final)	2 µl of 10 µM outside amplification primer #1 (OP-5’) 2 µl of 10 µM outside amplification primer #2 (OP-3’)
dNTP (0.2 mM final)	5 µl of 2 mM each (provided with KOD enzymes)
10X PCR buffer	5 µl (provided with KOD enzymes)
25 mM MgCl2	2 µl for KOD HiFi; none needed for XL
Sterile water	28 µl for KOD HiFi; 30 µl for XL
KOD HiFi enzyme for highest accuracy or KOD XL for TA cloning	0.4 µl KOD HiFi (Novagen) or 1 µl for KOD XL (Novagen)
Total	50 µl

Cycling conditions:

	KOD XL– up to 2 kb		KOD HiFi – up to 2 kb
25 cycles	94°C	30 sec	98°C	15 sec
	52°C	5 sec	52°C	2 sec
	72°C	30-60 sec/kb	72°C	20 sec
1 cycle	74°C	10 min

Analyze 5µl on agarose gel.
Purify remainder using Qiagen PCR purification columns, digest overnight, then gel purify. Ligate to vector of choice.

See Sample Results

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Common Stock Solutions

10 M Ammonium Acetate

To prepare a 10 M solution in 100 ml, dissolve 77 g of ammonium acetate in 70 ml of H₂O at room temperature. To prepare a 5 M solution in 100 ml, dissolve 38.5 g in 70 ml of H₂O. Adjust the volume to 100 ml with H₂O. Sterilize the solution by passing it through a 0.22μm filter. Store the solution in tightly sealed bottles at 4 ° C or at room temperature. Ammonium acetate decomposes in hot H₂O and solutions containing it should not be autoclaved.

Ampicillin

Prepare a stock of 100 mg/ml in water. Sterilize by filtration. Store at -20°C but avoid repeated freeze/thaw cycles. Use at a final concentration of 100 µg/ml.

Cresol Red Loading Dye - 2.5X - for PCR reactions

1M sucrose, 0.02% cresol. Prepare 1% cresol red in water (0.5 g/50 ml). To prepare loading dye, dissolve 17 g sucrose in a total volume of 49 ml of water. Add 1 ml 1% cresol red.

EB buffer (Qiagen recipe)

10 mM Tris-Cl pH 8.3

EDTA stock

To prepare 1 liter, 0.5M EDTA pH 8.0: Add 186.1 g of disodium EDTA-2H 2O to 800 ml of H 2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH. For tetrasodium EDTA, use 226.1 g of EDTA and adjust pH with HCl.

6x gel loading buffer

0.25% Bromophenol blue

0.25%Xylene cyanol FF

15% Ficoll Type 4000

120 mM EDTA

IPTG

IPTG is isopropylthio-b-D-galactoside. Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG in 8 ml of distilled H 2O. Adjust the volume of the solution to 10 ml with H 2O and sterilize by passing it through a 0.22μm disposable filter. Dispense the solution into 1 ml aliquots and store them at -20 ° C.

LB Medium

To make 1 liter, use 10 g tryptone, 5 g yeast extract, 10 g NaCl. Adjust pH to 7.0. Sterilize by autoclaving.

LB Agar

Dispense 15 g per liter of agar directly into final vessel. Prepare LB medium as above and add to agar. NOTE: Agar will not go into solution until it is autoclaved (or boiled). If adding antibiotics, autoclave medium first and allow to cool until warm to the touch, then add the antibiotic. Dispense about 30 ml per plate. Allow plates to dry either at 37°C overnight or 20 minutes in a laminar flow hood (lids removed). Store in original Petri plate bags, inverted, at 4°C for up to 2 weeks.

NaCl

To prepare 1 liter of a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H 2O. Adjust the volume to 1 liter with H 2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at room temperature.

NaOH

The preparation of 10 N NaOH involves a highly exothermic reaction, which can cause breakage of glass containers. Prepare this solution with extreme care in plastic beakers. To 800 ml of H2O, slowly add 400g of NaOH pellets, stirring continuously. As an added precaution, place the beaker on ice. When the pellets have dissolved completely, adjust the volume to 1 liter with H2O. Store the solution in a plastic container at room temperature. Sterilization is not necessary.

20X SB (electrophoresis buffer)

(Buffer diluted to 1X should be 10 mM Sodium hydroxide and pH 8.5 )

for 1 liter, weigh out 8 g NaOH and ~40 g boric acid - add water, dissolve and add additional boric acid until pH = 8.0; bring final volume to 1 liter.

SDS stock

10% or 20% (w/v) SDS. Also called sodium lauryl (or dodecyl) sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of electrophoresis-grade SDS in 900 ml of H₂O. Heat to 68 ° C and stir with a magnetic stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of concentrated HCl. Adjust the volume to 1 liter with H 2O. Store at room temperature. Sterilization is not necessary. Do not autoclave.

Use a mask when weighing this out.

20x SSC

0.3M Na(3) citrate

3M NaCl

SOB Medium: per liter:

Bacto-tryptone 20 g

Yeast extract 5 g

NaCl 0.584 g

KCl 0.186 g Mix components and adjust pH to 7.0 with NaOH and autoclave.

2 M Mg ++ stock:

MgCl 2-6H2O 20.33 g

MgSO 4 -7H2O 24.65 g

Distilled water to 100 ml. Autoclave or filter sterilize.

2 M Glucose

Glucose 36.04 g

Distilled water to 100 ml. Filter sterilize.

For SOB Medium + magnesium: Add 1 ml of 2 M Mg ++ stock to 99 ml SOB Medium.

For SOCMedium: Add 1 ml of 2 M Mg ++ stock and 1 ml of 2 M Glucose to 98 ml of SOB Medium.

3M Sodium Acetate - pH 5.2

To prepare a 3 M solution: Dissolve 408.3 g of sodium acetate-3H₂O in 800 ml of H 2O. Adjust the pH to 5.2 with glacial acetic acid. Adjust the volume to 1 liter with H₂O. Dispense into aliquots and sterilize by autoclaving.

Southern Solutions:

Depurination solution (for Southern blotting)

250mM HCl

Denaturation solution (for Southern blotting)

1.5M NaCl

0.5M NaOH

Neutralization solution (for Southern blotting)

1.5M NaCl

0.5M Tris-HCl, pH adjusted to 7.5

50x TAE

Prepare a 50x stock solution in 1 liter of H₂O:

242 g of Tris base

57.1 ml of glacial acetic acid

100 ml of 0.5 M EDTA (pH 8.0)

The 1x working solution is 40 mM Tris-acetate/1 mM EDTA.

5X (or 10X) TBE

Prepare a 5x stock solution in 1 liter of H2O:

54 g of Tris base

27.5 g of boric acid

20 ml of 0.5 M EDTA (pH 8.0)

The pH of the concentrated stock buffer should be approx. 8.3.. Some investigators prefer to use more concentrated stock solutions of TBE (10x as opposed to 5x). However, 5x stock solution is more stable because the solutes do not precipitate during storage. Passing the 5x or 10x buffer stocks through a 0.22μm filter can prevent or delay formation of precipitates.

TE buffer:

10 mM Tris-Cl (pH, usually 7.6 or 8.0)

1 mM EDTA (pH 8.0)

Use concentrated stock solutions to prepare. If sterile water and sterile stocks are used, there is no need to autoclave. Otherwise, sterilize solutions by autoclaving for 20 minutes. Store the buffer at room temperature.

1 M Tris-Cl – used at various pHs

Using Tris base : To make 1 liter, dissolve 121 g Tris Base in 800 ml of water. Adjust pH to the desired value by adding approximately the following:

pH = 7.4 about 70 ml of concentrated HCl

pH = 7.6 about 60 ml of concentrated HCl

pH = 8.0 about 42 ml of concentrated HCl

Make sure solution is at room temperature before making final pH adjustments. Bring final volume to 1 liter. Sterilize by autoclaving.

Using Trizma tables: an alternate procedure for preparing Tris solutions is to combine the proper amount of Tris Base and Tris Hydrochloride to achieve the desired value using Sigma's Tris tables.

WB (10% redistilled glycerol, 90% distilled water, v/v)

In a 1-liter graduated cylinder, add 100 ml of glycerol and 900 ml of distilled water. Cover with parafilm and mix thoroughly. Sterilized by autoclaving, and chill to 4°C.

Western Blotting Solutions:

1X Transfer buffer 1: 25 mM Tris, 192 mM Glycine, pH 8.3

Mix 3.03 g Tris, 14.4 g glycine; add dd water to 1 liter – do not adjust pH.

1X Transfer buffer 2: 25 mM Tris, 192 mM Glycine, pH 8.3, 20 % methanol

Mix 3.03 g Tris, 14.4 g glycine; add 200 ml methanol; add dd water to 1 liter – do not adjust pH. (NOTE: methanol is not needed for PVDF membranes)

10X Western Buffer: 200 mM Tris pH = 7.5; 1.5 M NaCl

To prepare 1X Western Buffer, dilute 10X buffer to 1X, adding Tween-20 to 0.1%. Remove 50 ml and set aside for the last two washes. To the remainder, add I-Block to 0.2%, heating gently with constant stirring until dissolved. Bring to room temperature before using.

X-gal 5-bromo-4-chloro-3-indolyl-b-D-galactoside (same recipe for X-phosphate)

Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. Use a glass or polypropylene tube. Wrap the tube containing the solution in aluminum foil to prevent damage by light and store at -20 ° C. It is not necessary to sterilize X-gal solutions.

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m6a

Extraction and purification of RNA - general

Eukaryotic RNAs

A single mammalian cell contains a total of approximately 1x10-5 µg of RNA :-

- 80 - 85% is ribosomal RNA (rRNA) 28, 18 and 5 s subunits,

- 15 - 20% is composed of a variety of low molecular weight species, transfer

RNA ( tRNA ), small nuclear RNAs etc.

- 1 - 5% is messenger RNA (mRNA).

rRNAs and tRNAs are of defined size and sequence and therefore relatively homogenous. These can be purified by gel electrophoresis or density gradient ultracentrifugation. mRNA is highly heterogeneous with respect to both size (~100 - 10 000 bases) and sequence except for the 3' terminus, which in eukaryotic cells usually carries a poly-adenylic acid (poly A) tract. The poly A tail is usually long enough to allow mRNAs to be purified by affinity chromatography on oligo(dT) cellulose.

RNA isolation

Successful isolation of RNA depends on the suppression of endogenous RNAses during cell / tissue lysis and avoiding contamination with exogenous RNAses during the isolation procedure.

1) Suppression of endogenous RNAses during cell / tissue lysis.

i) Tissues should be removed as fast as possible from the animal and then either homogenised immediately or snap frozen in liquid nitrogen / hexane on dry ice and stored at -70oC until required. Storage in ice-cold PBS until homogenisation is insufficient for most adult organs (RNAses are still very active)

ii) Simultaneous disruption of cells and inactivation of ribonucleases is achieved by lysis of the cells/tissues in strong denaturing agents :-

- 8M Urea - half life of RNase A is 3 minutes.

- 4M guanidinium hydrochloride - half life of RNase A is 10 seconds.

- 4M guanidinium isothiocyanate - 2.5x more effective on a molar basis

than guanidine HCl. Both anion and cation act as denaturants in the

GIT buffer but only the guanidine cation in GIT-HCl buffer.

iii) Inhibitors of RNAses.

RNasin. A protein isolated originally from human placenta (possibly a precursor of angiogenin). It inhibits RNAses A, B and C by non-competitive, non-covalent binding in equimolar ratio, with a Ki of 4 x 10-14M. **It requires a minimum DTT concentration of 1 mM ** (5 mM preferential) and has maximal activity in the pH range 7 - 8. Multiple freeze-thaw cycles and oxidising conditions will denature the protein and make it inactive. It has no activity against RNAses T1 or H, or those from aspergillus and S1 nuclease. It is of no use during the first stages of RNA isolation using strong denaturants, but should be included during the later stages of isolation, especially from pancreas, or if more gentle lysis methods are being used. 1 unit of RNasin reduces the activity of 5 ng of RNaseA by 50%.

Vanadyl-ribonucleoside complexes. Oxovanadium IV ions and any of the four ribonuleosides form complexes which bind to many RNAses and inhibit their activity almost completely. Used at a concentration of 10 mM during lysis of cells and subsequent isolation of RNA. Some enzymatic reactions are completely inhibited by even low levels of VRC eg. cell-free translation, whilst others are more tolerant eg. reverse transcription.

2) Avoiding contamination with exogenous RNAses during

isolation

i) DEPC - Diethyl pyrocarbonate - is used at 0.1% and is a strong but not absolute inhibitor of RNAses. Solutions and glassware are treated with DEPC solution, allowed to stand for several hours at room temperature and then autoclaved to remove the DEPC ( decomposes to water and carbon dioxide). Residual DEPC in a solution carboxylates purine residues in RNA (ie. A or G) : this causes low- efficiency translation in cell-free systems and will interfere with RNA:DNA / RNA:RNA hybrid formation if a large proportion of residues are modified. DEPC is a suspected carcinogen and unsuitable for Tris containing solutions.

ii) Equipment and solutions. Keep reagents, solutions, gel tanks etc. for RNA work only. Handle with gloves, treat with DEPC or any other RNase inhibitor and autoclave if possible. Use disposable gloves and change them frequently, and where possible, disposable plastic ware which is essentially RNase free.

Methods of RNA isolation

Four methods for direct isolation of RNAs are given below and each have their advantages and disadvantages. The relative merits of the two methods for selecting poly A+ RNA are given in m6e

m6b Isolation of total cellular RNA from mammalian cells using the

guanidinium isothiocyanate -ultracentrifugation method

Advantages

i) Total RNA is of high quality, reasonable yield and relatively pure from

protein or DNA contamination.

ii) Intact RNA from RNase-rich tissues such as pancreas can be consistently

isolated using this method.

iii) It is not as labour-intensive as the other methods (but still not suited to the preparation of large numbers of samples).

Disadvantages

i) It requires an ultracentrifuge.

ii) Large amounts of tissue need to be phenol-extracted to remove some of

the DNA and protein and then isopropanol precipitated before

centrifugation.

iii) Anecdotally, yields of poly A+ RNA are poorer from guanidinium-isolated

RNA, but I'm not sure I believe this.

References

i) V. Glisin, R. Crkvenjakov and C. Byus (1974) Ribonucleic acid isolated

by caesium chloride centrifugation Biochemistry 13:2633-7 (ref #470)

ii) A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter and

H. M. Goodman (1977) Rat insulin genes : construction of plasmids

containing the coding sequences Science 196:1313 (ref #237)

iii) J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter

(1979) Isolation of biologically active ribonucleic acid from sources

enriched in ribonuclease Biochemistry 18:5294-9 (ref #236)

iv) B. E. Faulkner-Jones, D. S. Cram, J. Kun and L. C. Harrison (1993)

Localisation and quantitation of expression of two glutamate

decarboxylase genes in pancreatic b-cells and other peripheral tissues of

mouse and rat Endocrinol 133:2962 - 2972 (ref #116)

m6c Isolation of total cellular RNA from mammalian cells using

guanidinium and phenol-chloroform

Advantages

i) Total RNA is of good quality and high yield.

ii) Intact RNA from RNase-rich tissues such as pancreas can be isolated

using a modified version of the protocol involving multiple phenol-

chloroform extractions and isopropanol precipitations.

iii) Large amounts of tissue / numbers of samples can be processed if

necessary.

Disadvantages

i) It is more labour-intensive than m6b

ii) The RNA from adult tissues is (in my hands) always contaminated with

DNA (and +/- protein).

Reference

P. Chomczynski and N. Sacchi (1987) Single-step method of RNA isolation by

acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 162:156-9

m6d Isolation of total cellular RNA using the lithium chloride - SDS -

urea method

Advantages

i) Large amounts of tissue / large numbers of samples can be processed.

ii) Total RNA is of good enough quality for RNase protection assays and for

some cultured cells and tissues it is good enough for Northern analysis.

RNase-rich tissues such as pancreas usually show minor degradation but

are occasionally completely degraded.

iii) Yields of total RNA are high.

Disadvantages

i) It is more labour-intensive than m6b

ii) The RNA from adult tissues is (in my hands) always contaminated with

DNA (and +/- protein), although less than with m6c

iii) RNA from some sources is not adequate for Northern analysis.

Reference

C. Auffray and F. Rougeon (1980) Purification of mouse immunoglobulin

heavy-chain messenger RNAs from total myeloma tumour RNA Eur J Biochem

107:303-314

m6f Direct isolation of poly A+ / mRNA from tissue lysates using

oligo(dT) cellulose

Advantages

i) Direct isolation of poly A+ RNA without isolating total RNA first.

ii) Yields of poly A+ selected RNA are reasonable.

iii) Relatively fast and allows large numbers of samples to be processed

simultaneously.

Disadvantages

i) Not suitable for some solid adult organs - RNA degrades partially or

completely.

ii) RNA is contaminated with DNA and some protein.

iii) RNA from some sources is not adequate for Northern analysis.

Reference

T. J. Gonda, D. K. Sheiness and J. M. Bishop (1982) Transcripts from the

cellular homologs of retroviral oncogenes : distribution among chicken tissues

Mol Cell Biol 2:617 - 624

table

Thesis notes from Chapter 3 Isolation of high quality, high purity RNA from adult organs rich in endogenous RNase

Large quantities of poly(A+) RNA was required to detect GAD mRNAs from most sources. The most rapid and widely used method for poly(A+) isolation by workers within the Institute was direct isolation from proteinase K digested tissue lysates (Reference #2 T. J. Gonda, D. K. Sheiness and J. M. Bishop (1982) Transcripts from the cellular homologs of retroviral oncogenes : distribution among chicken tissues Mol Cell Biol 2:617 - 624). This method produced RNA of good integrity from cell lines, but that from most organs was usually partially degraded (data not shown). Target mRNAs expressed at moderate to high levels, could be detected by RPA analysis of such RNA. Samples isolated from pancreas were invariably completely degraded.

Total cellular RNA was isolated before poly(A+) RNA purification by using a strong denaturant. A method using LiCl-urea lysis buffer (modified from Reference #4 C. Auffray and F. Rougeon (1980) Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumour RNA Eur J Biochem 107:303-314), allows the extraction of total RNA from large quantities of tissue. Despite the use of LiCl and acid phenol, the resulting RNA preparations were heavily contaminated with DNA and usually partially degraded (data not shown). Such samples were difficult to resuspend for RPA and gave a background ÔsmearÕ on the analytical gel.

The use of the more powerful guanidinium isothiocyanate (GIT) denaturant (Ref #236 J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294-9) forms the basis of several protocols for RNA extraction. Tissue architecture is disrupted and tissue RNAses inactivated simultaneously by GIT. Relatively small quantities of tissue are used. The GIT-acid phenol method (Reference #3 P. Chomczynski and N. Sacchi (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 162:156-9), produced initially intact but heavily DNA-contaminated samples. On resuspension in aqueous solution, RNA samples from pancreas degraded due to RNase contamination. Contaminating DNA and RNase were removed by ultracentrifugation of the GIT-tissue lysates through caesium chloride (Ref #236 J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294-9; Reference #237 A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter and H. M. Goodman (1977) Rat insulin genes : construction of plasmids containing the coding sequences Science 196:1313; Reference #470 Glisin, R. Crkvenjakov and C. Byus (1974) Ribonucleic acid isolated by caesium chloride centrifugation Biochemistry 13:2633-7). The resulting RNA was of high quality and purity. However, the ultracentrifugation took 21 hours and the caesium gradients were easily overloaded with tissue components resulting in contamination of the RNA pellet.

A protocol combining acid-phenol extraction of the tissue lysate with caesium ultracentrifugation was adopted. The organic extraction removed much of the protein and DNA from the lysate, allowing much larger quantities of tissue to be used for a single caesium gradient (Reference #116 B. E. Faulkner-Jones, D. S. Cram, J. Kun and L. C. Harrison (1993) Localisation and quantitation of expression of two glutamate decarboxylase genes in pancreatic b-cells and other peripheral tissues of mouse and rat Endocrinol 133:2962 - 2972)

Aqueous RNA solutions were protected from digestion by trace RNase contamination with RNasin. RNasin is denatured by SDS, and during the first phase of poly(A+) isolation using oligo(dT)cellulose, the RNA sample was vulnerable to hydrolysis. Proteinase K in SDS was used at 65oC to digest any residual RNase . Proteinase K is 12 times more active under these conditions than at room temperature (manufacturers specifications), and RNAses are partially inactivated by the high temperature and SDS.

In summary, poly(A+) RNA was routinely isolated from adult organs by the phenol-GIT-ultracentrifugation method followed by oligo(dT)cellulose chromatography. The direct isolation of poly(A+) RNA method was reserved for cell lines

m6bi

Isolation of total cellular RNA

using 4M guanidinium isothiocyanate lysis buffer and caesium chloride ultracentrifugation

Cultured cells or whole organs are homogenised in guanidinium isothiocyanate and the lysate is layered on to a dense caesium chloride cushion. The buoyant density of most RNAs in caesium chloride is much greater than that of other cellular components (> 1.8 g/ml). During ultracentrifugation, the RNA pellets at the bottom of the tube, the DNA bands in the caesium chloride cushion and the protein floats in the guanidinium lysis buffer. Small RNAs, eg 5s rRNA and tRNAs do not sediment well through CsCl.

The total RNA obtained is of very high quality, in good yield and is pure from protein and DNA contamination as long as the capacity of the gradient is not exceeded. Intact RNA from RNase-rich tissues such as pancreas can be consistently isolated using this method. Although not as labour-intensive as the other methods, it is still not suited to the preparation of large numbers of samples.

Protocol

General

Day 1 Homogenise tissues and assemble caesium chloride gradients

Day 2 Dissemble gradients, purify and quantitate RNA and check its integrity

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reactions.

Phosphate-buffered saline Guanidinium lysis buffer :- 4M Guanidinium isothiocyanate, 25 mM sodium acetate pH 6.0 and 1 mM EDTA pH 8.0. Store at room temperature - it will keep for months but it is light sensitive 5.7M caesium chloride, 25 mM acetate pH 6.0, 1 mM EDTA, 0.1% DEPC. Autoclave and store at room temperature 20% (w/v) N-lauryl sarcosine 10 M b-mercaptoethanol Sterile water 100 mM DTT RNasin RNase inhibitor (Promega) Water-saturated acid phenol Chloroform 8M LiCl

Equipment

Polytron or equivalent

Ultracentrifuge with swing-out rotor

Polyallomer 13 ml ultracentrifuge tubes (autoclaved if desired)

Methods

In advance

1 Clean Polytron probe with 3 changes of distilled water. If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA

Day 1

2 Harvest tissues or cells for RNA isolation. Tissues Killed animals by either cervical dislocation or by carbon dioxide asphyxia. Remove tissues immediately and either - homogenise in 7 ml of GIT lysis buffer with a Polytron or equivalent, or - snap-freeze in a liquid hexane bath cooled on dry ice. Store snap-frozen tissues at -70oC until required for RNA isolation. NB do not store too long, especially if tissue is RNase rich eg pancreas Cells Wash adherent cell cultures with ice cold PBS, and then completely lyse in the culture flask with 7 ml GIT lysis buffer / 108 to 109 cells. Draw the lysate several times through an 18 gauge needle with a 20 ml syringe to shear genomic DNA. The lysate may also be sheared using the Polytron Number of adult organs per gradient (ie 7 ml of GIT lysis buffer) These figures are for young (~ 12 week) female mice. Less tissue can be processed from older and / or male mice. 3 x brains >3 x thymus 4 x lungs 3 x hearts >6 x eyes 1 x spleen 1 x pancreas 2 x kidneys 4 x testes Small piece of liver If more tissue than this is required, then use the method described in m6biii 1 3 Make a 4 ml 5.7M CsCl cushion in the bottom of each polyallomer ultracentrifuge tube - remove all bubbles with a sterile pipette tip 4 Add 1 ml of 5.7 M CsCl, 1 mM EDTA, 4 x 30 ml drops of 10 M b-mercaptoethanol and 8 x 30 ml drops of 20% (w/v) N-lauryl sarcosine to each GIT-tissue lysate 2. Mix and then layer CsCl-GIT lysate over the CsCl cushion. 5 Balance ultracentrifuge tubes to within 0.05 g and centrifuge at 33 000 rpm (180 000 g at the tube bottom in the SW40Ti rotor) for 21 hours 3 at 20oC 4

Day 2

6 Remove the GIT-CsCl by placing a pipette at the air-liquid interface and gently aspirating the tube contents. Swab the inside of the tube to remove residual GIT-CsCl, taking care not to touch the clear, gelatinous RNA pellet at the tube bottom 7 Resuspend the RNA pellet in 400 - 750 ml of 4M GIT, 25 mM Na Acetate pH 6.0 by gently aspirating up and down with a wide bore pipette. 8 Extract the resuspended RNA with and equal volume of acid-phenol 5 until the interface is clear 9 Extract once with chloroform 10 Precipitate with 1 volume of isopropanol and 1 / 10 volume of 8M LiCl 6 and recover by centrifugation at 1200g for 10 minutes 11 Wash the pellet in 70% ethanol and then resuspend in 50 - 500 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water. 12 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm. 13 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis

14 Store the RNA at -70oC

Notes

1 This method should give intact, pure total RNA. Both 28 and 18 s rRNA bands are clearly seen. The 5 s rRNA can be seen only if CsCl is added to the GIT-tissue lysate

2 Tissue which contain fat - eg brain or other components which may block the gradient - eg thyroidal colloid, should either be processed using the protocol in m6biii, or spun at 1 200 g to remove tissue debris / fat / colloid before layering over the CsCl cushion

3 Liver and kidney RNA preparations are often glycogen contaminated

4 The ratio of the 260 : 280 UV absorbance readings (should be > 2.0 for clean RNA) is often poor for GIT-isolated tissues and the UV absorbance at 260 nm may bear little relationship to the amount of RNA present when checked by gel electrophoresis. Contaminating trace amounts of GIT or phenol interfere with UV absorbance by RNA at these wavelengths

References

Reference #143 Tan Lab Library 07-94> Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular Cloning, A Laboratory Manual, Second Edition. Cold Spring Harbour Laboratory Press. Reference #151 Tan Lab Library 07-94> Davis LG, Dibner MD, Battey JF. 1986 Basic Methods in Molecular Biology. Elsevier. New York Reference #237 Tan Lab Library 07-94>

A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter and H. M. Goodman (1977) Rat insulin genes : construction of plasmids containing the coding sequences Science 196:1313 -

Reference #236 Tan Lab Library 07-94>

J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294-9

Reference #470 Tan Lab Library 07-94>

V. Glisin, R. Crkvenjakov and C. Byus (1974) Ribonucleic acid isolated by cesium chloride centrifugation Biochemistry 13:2633-7

Reference #166 Tan Lab Library 07-94>

B. E. Faulkner-Jones, D. S. Cram, J. Kun and L. C. Harrison (1993) Localisation and quantitation of expression of two glutamate decarboxylase genes in pancreatic b-cells and other peripheral tissues of mouse and rat Endocrinol 133:2962 - 2972

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m6bii

Isolation of total cellular RNA using 4M guanidinium isothiocyanate lysis buffer and caesium chloride ultracentrifugation :

Mini-ultracentrifuge method

Cultured cells, small pieces of tissue or embryos are homogenised in guanidinium isothiocyanate and the lysate is layered on to a dense caesium chloride cushion. The buoyant density of most RNAs in caesium chloride is much greater than that of other cellular components (> 1.8 g/ml). During ultracentrifugation, the RNA pellets at the bottom of the tube, the DNA bands in the caesium chloride cushion and the protein floats in the guanidinium lysis buffer. Small RNAs, eg 5s rRNA and tRNAs do not sediment well through CsCl.

Protocol

General

Day 1 Homogenise tissues and assemble caesium chloride gradients

Day 2 Dissemble gradients, purify and quantitate RNA and check its integrity

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reactions.

Equipment

Polytron or equivalent

Ultracentrifuge with swing-out rotor

Polyallomer 2.2 ml ultracentrifuge tubes (autoclaved if desired)

Methods

In advance

1 Clean Polytron probe with 3 changes of distilled water. If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA

Day 1

2 Harvest tissues or cells for RNA isolation. Tissues Only small pieces of tissue or embryos can be used : whole adult organs will exceed the capacity of the gradients unless phenol : chloroform extracted first. Remove tissues or embryos immediately and either - homogenised in 5 ml of GIT lysis buffer with a Polytron or equivalent, or - snap-freeze in a liquid hexane bath cooled on dry ice. Store snap-frozen tissues at -70oC until required for RNA isolation. NB do not store too long, especially if tissue is RNase rich eg pancreas Cells Wash adherent cell cultures with ice cold PBS, and then completely lyse in the culture flask with 5 ml GIT lysis buffer / 106 to 107 cells. Draw the lysate several times through an 18 gauge needle with a 20 ml syringe to shear genomic DNA. The lysate may also be sheared using the Polytron 3 Precipitate crude nucleic acids with 1 volume of isopropanol and 1 / 10 volumes of 8M LiCl and recover by centrifugation 4 Resuspended in 1 ml of 4M GIT lysis buffer. Remove insoluble material from the cell and tissue lysates by centrifugation. 5 Make a 600 ml 5.7M CsCl cushion in the bottom of each ultracentrifuge tube - remove all bubbles with a sterile pipette tip 4 Add 100 ml of 5.7M CsCl, 1 mM EDTA, 30 ml of 10 M b-mercaptoethanol and 30 ml of 20% (w/v) N-lauryl sarcosine to each GIT-tissue lysate 1. Mix and then layer CsCl-GIT lysate over the 600 ml CsCl cushion. 5 Balance ultracentrifuge tubes to within 0.05 g and centrifuge at 45 000 rpm (180 000 g at the tube bottom in the TLS-55 rotor in a Beckman TL-100 ultracentrifuge) for 6 - 8 hours 2 at 20oC 3.

'Day 2'

6 Remove the GIT-CsCl by placing a pipette at the air-liquid interface and gently aspirating the tube contents. Swab the inside of the tube to remove residual GIT-CsCl, taking care not to touch the clear, gelatinous RNA pellet at the tube bottom 7 Resuspend the RNA pellet in 100 ml of 4M GIT, 25 mM Na Acetate pH 6.0 by gently aspirating up and down with a wide bore pipette. 8 Extract the resuspended RNA with and equal volume of acid-phenol 4 until the interface is clear 9 Extract once with chloroform 10 Precipitate with 1 volume of isopropanol and 1 / 10 volume of 8M LiCl 5 and recover by centrifugation at 1200g for 10 minutes 11 Wash the pellet in 70% ethanol and then resuspend in 20 - 100 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water. 12 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm. 13 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis

14 Store the RNA at -70oC

Notes

1 This method should give intact, pure total RNA. Both 28 and 18 s rRNA bands are clearly seen. The 5 s rRNA can be seen only if CsCl is added to the GIT-tissue lysate

2 The ratio of the 260 : 280 UV absorbance readings (should be > 2.0 for clean RNA) is often poor for GIT-isolated tissues and the UV absorbance at 260 nm may bear little relationship to the amount of RNA present when checked by gel electrophoresis. Contaminating trace amounts of GIT or phenol interfere with UV absorbance by RNA at these wavelengths

2 After resuspending the RNA pellet in GIT - steps 4 or 7 - the solution may be frozen at -70oC prior to further purification

References

A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter and H. M. Goodman (1977) Rat insulin genes : construction of plasmids containing the coding sequences Science 196:1313 -

Reference #236 Tan Lab Library 07-94>

J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294-9

Reference #470 Tan Lab Library 07-94>

V. Glisin, R. Crkvenjakov and C. Byus (1974) Ribonucleic acid isolated by cesium chloride centrifugation Biochemistry 13:2633-7

Reference #166 Tan Lab Library 07-94>

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This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95.

1 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 2 If the RNA yield is expected to be high, the rotor can be stopped after 6 hours 3 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 4 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 5 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (4) and (5) reduce the amount of contamination

m6biii

Isolation of total cellular RNA using 4M guanidinium isothiocyanate lysis buffer and caesium chloride

ultracentrifugation : Large scale isolation

of total cellular RNA from adult organs

Whole organs are homogenised in guanidinium isothiocyanate and then phenol-chloroform extracted to remove much of the protein and DNA content of the lysate. The partially purified lysate is then layered on to a dense caesium chloride cushion. The buoyant density of most RNAs in caesium chloride is much greater than that of other cellular components (> 1.8 g/ml). During ultracentrifugation, the RNA pellets at the bottom of the tube, the DNA bands in the caesium chloride cushion and the protein floats in the guanidinium lysis buffer. Small RNAs, eg 5s rRNA and tRNAs do not sediment well through CsCl.

Large amounts of very high quality total RNA in good yield can be obtained from adult organs. It is free of protein and DNA contamination. Intact RNA from RNase-rich tissues such as pancreas can be consistently isolated using this method. Although not as labour-intensive as the other methods, it is still not suited to the preparation of large numbers of samples.

Protocol

General

Day 1 Homogenise tissues and assemble caesium chloride gradients

Day 2 Dissemble gradients, purify and quantitate RNA and check its integrity

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reactions.

Guanidinium lysis buffer :- 4M Guanidinium isothiocyanate, 25 mM sodium acetate pH 6.0 and 1 mM EDTA pH 8.0. Store at room temperature - it will keep for months but it is light sensitive 5.7M caesium chloride, 25 mM acetate pH 6.0, 1 mM EDTA, 0.1% DEPC. Autoclave and store at room temperature 20% (w/v) N-lauryl sarcosine 10 M b-mercaptoethanol Sterile water 100 mM DTT RNasin RNase inhibitor (Promega) Water-saturated acid phenol Chloroform 8M LiCl

Equipment

Polytron or equivalent

Ultracentrifuge with swing-out rotor

Polyallomer 13 ml ultracentrifuge tubes (autoclaved if desired)

Methods

In advance

1 Clean Polytron probe with 3 changes of distilled water. If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA

Day 1

2 Harvest tissues or cells for RNA isolation. Killed animals by either cervical dislocation or by carbon dioxide asphyxia. Remove tissues immediately and either - homogenise in up to 25 ml of GIT lysis buffer with a Polytron or equivalent, or - snap-freeze in a liquid hexane bath cooled on dry ice. Store snap-frozen tissues at -70oC until required for RNA isolation. NB do not store too long, especially if tissue is RNase rich eg pancreas 3 Add 0.5 volumes of acid-phenol, vortex, and then phase-separate by the addition of 0.5 volumes of chloroform 1. Vortex and recover aqueous phase by centrifugation. 4 Repeat the phenol-chloroform extractions until the interface is clear. 5 Precipitate crude nucleic acids with 1 volume of isopropanol and 1 / 10 volume of 8M LiCl. Recover by centrifugation. 6 Wash the pellet in 70% ethanol and then resuspend in 7 ml of 4M GIT 7 Make a 4 ml 5.7M CsCl cushion in the bottom of each polyallomer ultracentrifuge tube - remove all bubbles with a sterile pipette tip 8 Add 1 ml of 5.7 M CsCl, 1 mM EDTA, 4 x 30 ml drops of 10 M b-mercaptoethanol and 8 x 30 ml drops of 20% (w/v) N-lauryl sarcosine to each GIT-tissue lysate 2. Mix and then layer CsCl-GIT lysate over the CsCl cushion. 9 Balance ultracentrifuge tubes to within 0.05 g and centrifuge at 33 000 rpm (180 000 g at the tube bottom in the SW40Ti rotor) for 21 hours 3 at 20oC 4

Day 2

10 Remove the GIT-CsCl by placing a pipette at the air-liquid interface and gently aspirating the tube contents. Swab the inside of the tube to remove residual GIT-CsCl, taking care not to touch the clear, gelatinous RNA pellet at the tube bottom 11 Resuspend the RNA pellet in 400 - 750 ml of 4M GIT, 25 mM Na Acetate pH 6.0 by gently aspirating up and down with a wide bore pipette. 12 Extract the resuspended RNA with and equal volume of acid-phenol 5 until the interface is clear 13 Extract once with chloroform 14 Precipitate with 1 volume of isopropanol and 1 / 10 volume of 8M LiCl 6 and recover by centrifugation at 1200g for 10 minutes 15 Wash the pellet in 70% ethanol and then resuspend in 50 - 500 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water. 16 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm. 17 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis

18 Store the RNA at -70oC

Notes

1 This method should give intact, pure total RNA. Both 28 and 18 s rRNA bands are clearly seen. The 5 s rRNA can be seen only if CsCl is added to the GIT-tissue lysate

References

A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter and H. M. Goodman (1977) Rat insulin genes : construction of plasmids containing the coding sequences Science 196:1313 -

Reference #236 Tan Lab Library 07-94>

J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294-9

Reference #470 Tan Lab Library 07-94>

V. Glisin, R. Crkvenjakov and C. Byus (1974) Ribonucleic acid isolated by cesium chloride centrifugation Biochemistry 13:2633-7

Reference #166 Tan Lab Library 07-94>

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This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95.

1 Phenol and GIT are miscible. Chloroform must be added to searate the two phases. Heating the GIT-phenol solution to 65oC increases the efficiency of the organic solvent extraction steps 2 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 3 If the RNA yield is expected to be high, the rotor can be stopped after 16 - 18 hours 4 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 5 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 6 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination

m6biii

Isolation of total cellular RNA using 4M guanidinium isothiocyanate lysis buffer and caesium chloride

ultracentrifugation : Large scale isolation

of total cellular RNA from adult organs

Protocol

General

Day 1 Homogenise tissues and assemble caesium chloride gradients

Day 2 Dissemble gradients, purify and quantitate RNA and check its integrity

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reactions.

Equipment

Polytron or equivalent

Ultracentrifuge with swing-out rotor

Polyallomer 13 ml ultracentrifuge tubes (autoclaved if desired)

Methods

In advance

1 Clean Polytron probe with 3 changes of distilled water. If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA

Day 1

Day 2

18 Store the RNA at -70oC

Notes

1 This method should give intact, pure total RNA. Both 28 and 18 s rRNA bands are clearly seen. The 5 s rRNA can be seen only if CsCl is added to the GIT-tissue lysate

References

A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter and H. M. Goodman (1977) Rat insulin genes : construction of plasmids containing the coding sequences Science 196:1313 -

Reference #236 Tan Lab Library 07-94>

J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294-9

Reference #470 Tan Lab Library 07-94>

V. Glisin, R. Crkvenjakov and C. Byus (1974) Ribonucleic acid isolated by cesium chloride centrifugation Biochemistry 13:2633-7

Reference #166 Tan Lab Library 07-94>

[Previous] [Top] [Next]

This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95.

m6c

Isolation of total cellular RNA

from mammalian cells using guanidinium and phenol-chloroform

This method can be used for some adult tissues, most cell lines and embryos. The protocol is a modified version of that described by Chomczynski and Sacchi. Harvested tissues are homogenised in guanidinium and then phenol-chloroform extracted to remove contaminating proteins. The use of acid phenol also selectively removes DNA. Provided the initial tissue source is not too rich in RNase, the resulting RNA is usually largely intact, but it is rarely as free of DNA as that obtained from caesium chloride gradients. It is suited to the preparation of large numbers of small samples for RT-PCR screening

Protocol

General

Harvest and homogenise tissues - 1 - 4 hours

Isolate and purify RNA - ~ 4 hours

Formaldehyde-agarose gel check recovered RNA - ~ 4 hours

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reactions.

Phosphate-buffered saline Guanidinium lysis buffer :- 4M Guanidinium isothiocyanate, 25 mM sodium acetate pH 6.0 and 1 mM EDTA pH 8.0. Store at room temperature - it will keep for months but it is light sensitive 20% (w/v) N-lauryl sarcosine 10 M b-mercaptoethanol Sterile water 100 mM DTT RNasin RNase inhibitor (Promega) Water-saturated acid phenol Chloroform 8M LiCl

Equipment

Polytron or equivalent

Methods

1 Clean Polytron probe with 3 changes of distilled water. If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA 2 Harvest tissues or cells for RNA isolation. Tissues Killed animals by either cervical dislocation or by carbon dioxide asphyxia. Remove tissues immediately and either - homogenise in up to 25 ml of GIT lysis buffer in a 50 ml tube with a Polytron or equivalent, or - snap-freeze in a liquid hexane bath cooled on dry ice. Store snap-frozen tissues at -70oC until required for RNA isolation. NB do not store too long, especially if tissue is RNase rich eg pancreas1,2,3 Cells Wash adherent cell cultures with ice cold PBS, and then completely lyse in the culture flask with 7 ml GIT lysis buffer / 108 to 109 cells. Draw the lysate several times through an 18 gauge needle with a 20 ml syringe to shear genomic DNA. The lysate may also be sheared using the Polytron 3 Add N-lauryl sarcosine and b-mercaptoethanol to final concentrations of 0.5% and 100 mM, respectively 4 Add 0.5 volumes of acid phenol 4 to the tissue lysate and vortex vigorously. NB GIT and phenol are miscible (GIT and phenol mixed = the commercial RNasol preparation). Particularly if the DNA content of the tissue is high (spleen, thymus) heat the tube contents to 65oC for 15 minutes and vortex intermittently 5 Add 0.5 volumes of chloroform, vortex and then separate the two phases by centrifugation. Recover the upper aqueous phase and repeat the extraction until the interface is clear 6 Precipitate the RNA with one volume of isopropanol and 0.1 volumes of lithium chloride 5 and recover by centrifugation at 1200g for 10 minutes 7 Wash the pellet in 70% ethanol and then resuspend in 50 - 500 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water6 8 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm. 9 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis

10 Store the RNA at -70oC

Notes

The ratio of the 260 : 280 UV absorbance readings (should be > 2.0 for clean RNA) is often poor for GIT-isolated tissues and the UV absorbance at 260 nm may bear little relationship to the amount of RNA present when checked by gel electrophoresis. Contaminating trace amounts of GIT or phenol interfere with UV absorbance by RNA at these wavelengths

References

Reference #237 A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter and H. M. Goodman (1977) Rat insulin genes : construction of plasmids containing the coding sequences Science 196:1313 -

Ref #236 J. M. Chirgwin, A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease Biochemistry 18:5294-9

Reference #143 J. Sambrook, E. F. Fritsch and T. Maniatis (1989) Molecular Cloning, A Laboratory Manual.

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1 Usually it is possible to use 5 ml of less GIT buffer 2 If small amounts of tissue are being harvested and the appropriate homogeniser probe is available, then use an Eppendorf tube and 0.5 - 1 ml of GIT buffer 3 Very small tissue fragments can be dissolved directly in 0.5 ml of GIT at 37oC 4 RNA partitions into the aqueous phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 5 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 6 The pellet may be resuspended in GIT, and the phenol-chloroform extractions repeated a second or even third time. This is necessary for pancreas

m6d

Isolation of total cellular RNA using

the lithium chloride - SDS - urea method

(Auffray-Rougeon).

This method can be used for some adult tissues, most cell lines and embryos. The protocol is a modified version of that described by Auffray and Rougeon. Harvested tissues are homogenised in urea-lithium and the relatively insoluble lithium-RNA salts are precipitated overnight at 4oC. Much of the DNA remains in solution. The recovered lithium-RNA is acid phenol-chloroform extracted to remove contaminating proteins. The use of acid phenol also selectively removes DNA. Urea is a slower denaturant than guanidinium, but provided the initial tissue source is not too rich in RNase, the resulting RNA is usually largely intact, but it is always DNA contaminated. The yields from this method are good

Protocol

General

Day 1 Harvest and homogenise tissues. Precipitate overnight

Day 2 Isolate and purify RNA

Day 3 Formaldehyde-agarose gel check recovered RNA

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reaction

Phosphate-buffered saline 6M urea, 3M lithium chloride, 0.5% SDS lysis buffer. Store at 4oC 3M sodium acetate pH 6.0 20% SDS in sterile water Sterile water 100 mM DTT RNasin RNase inhibitor (Promega) Water-saturated acid phenol Chloroform 8M LiCl

Equipment

Polytron or equivalent

Sorval centrifuge and sterile glass (Corex) tubes for recovery of RNA

Methods

In advance

1 Clean Polytron probe with 3 changes of distilled water. If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA

Day 1

2 Harvest tissues or cells for RNA isolation. Kill the animals by either cervical dislocation or by carbon dioxide asphyxia. Remove the tissues immediately and either Homogenise in lithium-urea lysis buffer1 with a Polytron or equivalent, or Snap-freeze in a liquid hexane bath cooled on dry ice and store snap-frozen tissues at -70oC until required for RNA isolation. NB do not store too long, especially if tissue is RNase rich eg pancreas Wash adherent cell cultures with ice cold PBS, and then completely lyse in the culture flask with 10 ml of lysis buffer / 108 to 109 cells. Draw the lysate several times through an 18 gauge needle with a 20 ml syringe to shear genomic DNA. The lysate may also be sheared using the Polytron 3 Add SDS to a final concentration of 0.5% 4 Transfer the lysates to sterile 30 ml Corex tubes and leave at 4oC overnight

Day 2

5 Recover the RNA by centrifugation at 1 000 g for 30 minutes at 4oC2. Discard the supernatant and fully resuspend the pellet in 10 ml of 10 mM sodium acetate and 0.5% SDS 6 Extract the solution twice with acid-phenol 3 and once with acid phenol-chloroform 7 Precipitate the RNA with one volume of isopropanol and 0.1 volumes of lithium chloride 4 and recover by centrifugation at 1200g for 10 minutes 8 Wash the pellet in 70% ethanol and then resuspend in 50 - 500 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water5 9 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm

Day 3

10 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis

11 Store the RNA at -70oC

Notes

The ratio of the 260 : 280 UV absorbance readings (should be > 2.0 for clean RNA) may be poor for lithium-urea-phenol isolated tissues and the UV absorbance at 260 nm may bear little relationship to the amount of RNA present when checked by gel electrophoresis. Contaminating trace amounts of phenol interfere with UV absorbance by RNA at these wavelengths

References

Reference #4 C. Auffray and F. Rougeon (1980) Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumour RNA Eur J Biochem 107:303-314

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1 Use 10 ml of lysis buffer per gram of tissue 2 Prolonged centrifugation makes the pellet very difficult to resuspend 3 RNA partitions into the aqueous phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 4 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 5 The pellet may be resuspended in lithium-urea, and the phenol-chloroform extractions repeated a second or even third time. This is necessary for pancreas

m7dii

RNase Protection Assay - Protocol

This method can be used to detect and quantitate mRNA, to map mRNA termini and to determine the position of introns within the corresponding gene.

High specific activity 32P-UTP-labelled single-stranded cRNA is generated and purified, then hybridised in excess in solution to the target mRNA : this ensures that all / most of the target mRNA is hybridised to the cRNA probe. A combination of RNaseA and RNaseT1 are used to digest unhybridised, single-stranded RNA and the digestion products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography / PhosphorImaging. The undigested cRNA probe will contain a stretch of plasmid sequence and is therefore larger than the mRNA which it "protects" from the action of RNase by duplex formation. It will migrate slower than the protected fragments and is used for their identification

Protocol

Reagents - make up all reagents in sterile MilliQ water

Reagents for in vitro transcription - see section on In vitro transcription Target RNA Formamide deionise with BioRad mixed bed resin for 1 hour at room temperature. Check the pH - it should be neutral or less than 7.4. If it is greater, then discard that batch of formamide. Store deionised formamide in aliquots at -20oC1 10 x RNase protection buffer 400 mM PIPES (disodium piperazine-N,N'-bis[2-ethansulphonic acid]),10 mM EDTA and 4 M NaCl, pH 6.4. Add PIPES as the free acid (RMM 302, Boehringer 239 496) and adjust pH to 6.4 : the PIPES will not go into solution at lower pH. Autoclave and store in aliquots at -20oC tRNA at 10 mg / ml in sterile water. Dissolve the tRNA in sterile water, extract twice against phenol:chloroform, ethanol precipitate, wash with 70% ethanol and then resuspend in sterile water. Store in aliquots at -20oC RNaseA 2 mg / ml of Sigma R5250 X-A in 10 mM Tris pH 7.5, 15 mM NaCl. Store small aliquots at -20oC RNase T1 100 mg / ml Sigma R1003 in 50% glycerol and 10 mM NaPO4 pH 6.5. Store small aliquots at -20oC. 10 x RNase digestion buffer 100 mM Tris pH 7.5, 50 mM EDTA, 3M NaCl. Autoclave to sterilise and store at room temperature 20% SDS in sterile water Freshly made proteinase K at 10 mg / ml in water2 Water-saturated acid phenol Chloroform Isopropanol Formamide loading buffer 90% deionised formamide, 1 x TBE and bromophenol blue. (Make 5 ml viscous enough to sink to the bottom of the well without diffusing upwards by adjusting the amount of saturated BPB solution added) Reagents and equipment for denaturing polyacrylamide gel electrophoresis of RNA

Methods - read the notes at the end of the protocol

BEFORE setting up the assay

In advance

1 Prepare template DNA for generation of the cRNA probe 2 Prepare all RNA samples, both the test samples and the positive and negative control 'cold' sense cRNAs

Day 1

3 Make high specific activity a32P-UTP-labelled cRNA. Use a 5 - 6% denaturing polyacrylamide preparative gel. It will take ~ 5 - 8 hours to transcribe, purify and elute the probe 4 Assemble the hybridisation reactions Make all the reactions up from a single 'master mix' to reduce variations between samples. Depending on the mount of RNA to be analysed3, and the amount of contaminating DNA in the sample4, use reaction volumes between 30 - and 60 ml. Use the PIPES buffer at a final concentration of 1 x and the formamide at 80%5 5 All reactions should contain the same amount of RNA in total - make up the difference between tubes with tRNA eg 20 or 50 mg per reaction6 6 Include the following controls :- Negative control : tRNA only. This will indicate if the RNAses are not working, if there is still template DNA in the probe preparation and whether the probe self-hybridises significantly Positive control : essential with an uncharacterised probe. Use synthetic sense cRNA7 or a known source of the 'natural' RNA. A plasmid will do if there is nothing else Positive control : a house-keeping gene mRNA to check the integrity of the mRNA eg GAP-DH. This can be performed in the same reaction - see the Notes section at the end 7 Make sure the RNA is completely dissolved8 - heat at 37oC if necessary - before adding the probe 8 Add 1 ml of the cRNA probe which must be in excess for accurate quantitation. The amount has to be determined empirically but as a rough rule of thumb, using a 200 - 400 base probe and a yellow b-counter, for a low abundance mRNA, 100 - 200 cps / ml is sufficient and for a high abundance mRNA 500 - 600 cps / ml is need 9 Vortex tube and then heat denature the reactions at 80oC for 2-5 minutes and transfer to a pre-warmed rack at 45oC9. Incubate at 45oC10 and allow to hybridise for at least 8 hours11

Day 2

11 Take the tubes out of the incubator and chill on ice Steps 11 - 23 will take most of the rest of the day 12 Immediately add 350 ml of ice cold RNase digestion solution, mix with the hybridisation reaction by vortexing and incubate for 30 minutes. A good starting point is 1/5x RNase ie. 40 ml of RNaseA solution and 40 ml of RNaseT1 solution in 10 ml of 1x RNase digestion buffer (ie 8 mg / ml RNaseA and 0.4 mg / ml RNaseT1), at 37oC12 13 Add 20 ml of 10% SDS and 10 ml of 10 mg / ml proteinase K solution and incubate at 37oC for a further 30 minutes13 14 Extract once with an equal volume of acid-phenol14 15 Remove the aqueous phase, add 20 mg of tRNA and precipitate with 600 ml of isopropanol 16 Mix the tube contents by vortexing and precipitate the digestion products on ice for 10 minutes 17 Recover the digestion products by centrifugation at room temperature for 20 minutes 18 Identify the pellet then aspirate all the isopropanol - do not lose the pellet 19 Wash the pellet extensively with 70% ethanol, re vortex, re centrifuge and aspirate as much ethanol as possible 20 Dry the pellet until all traces of ethanol are gone, and resuspend in 5 - 6 ml of formamide loading buffer15 21 Once the pellet is fully resuspended in loading buffer, denature the digestion products at 80oC for 2 minutes16 22 Chill the sample on ice 23 Analyse the digestion products by denaturing polyacrylamide gel electrophoresis and either autoradiography or PhosphorImaging. - Make the analytical gel with ~>2% more polyacrylamide than the preparative gel used for the cRNA probe. - Always run an aliquot of undigested probe - this will run behind the protected fragments and be an indication if the probe is hybridising to mRNA target or contaminating DNA in the test reactions - Allow a lane between the undigested probe sample and the test sample lanes if possible (eg use the tRNA negative control sample) - Calibrate the amount of undigested probe to load onto the gel. Use the hand-held b-counter to gauge the amount of radioactivity in the positive control and test samples. Load one aliquot that is equivalent to ~half the counts in the positive control sample, and one aliquot that is equivalent to ~half the counts in the test samples. Similar exposure times can then be used for undigested probe + positive control, and undigested probe and test sample(s) - The tRNA plus probe lane should be empty. High abundance mRNA will be visible within the hour. Low abundance (0.1 pg) mRNA will need 5 - 7 days with a PhosphorImager - Kodak or Fuji film give good, long exposure autoradiographs

Notes

1 The standard cRNA probe preparation is 11 ml volume containing 40 mM TrisHCl (pH 8.0), 6 mM MgCl2, 2 mM spermidine HCl, 10 mM NaCl, 10 mM DTT, 100 mg/ml BSA, 20 nM linearised phagemid template DNA, 250 mM ATP, 250 mM CTP, 250 mM GTP, 1 unit ml of RNasin, 500 - 1000 units / ml of T7 RNA polymerase, 5 ml of a32P-UTP (3000 Ci / mmol, 10 mCi/ ml) at a final concentration of 1.5 mM and unlabelled UTP at a final concentration of 10 mM 2 Hotter probes do not necessarily make more sensitive RPAs, and due to the greater radiolysis of a higher specific activity probe, they may be less sensitive, less aesthetic or both. A good compromise for specific activity of cRNA probes is 11.5 mM UTP - 1.5 mM hot and 10 mM cold - reasonable proportion of full length transcripts and a hot probe. The standard protocol above generates a theoretical maximum of 167 ng of cRNA with an SA. of 0.72x109 dpm/mg, which is 14% of the theoretical maximum SA. of 5.1x109 dpm/mg. 3 DNaseI treatment of transcription reactions prior to gel purification to remove the DNA template : template contamination will be a problem if you don't. Especially noticeable on longer exposures ® unaesthetic / uninterpretable results 4 Use the probes as soon as possible after gel purification and definitely within 24 hours. All the radiolysed fragments are complementary to the target and will protect partial fragments. The resulting RPA will be less aesthetic and less sensitive 5 Poly A+ RNA gives cleaner results than total RNA. Although you can use a large amount of RNA in a single reaction, it generates more sub bands and a less aesthetic RPA. Any mistakes during the course of the assay will be compounded and be even more apparent than usual! 6 I have used both 'dried down' and precipitated RNA - I don't really think that there is a difference between the two methods as long as the RNA is fully resuspended before the experiment starts. If drying down RNA, don't completely desiccate it - it will be very difficult to get into solution. If the RNA has been precipitated, wash out the salt with 70% ethanol before resuspension 7 To determine the amount of probe needed to produce acceptable results, do a series of reactions with varying ratios of probe to (synthetic) target mRNA to find the amount of probe giving the best signal : noise ratio. 8 Determine the amount of target mRNA needed to produce acceptable results by doing a series with the appropriate amount of probe. 9 If using a house-keeping gene as a positive control, it can be done in the same reaction tube as the test probe as long as the protected fragment of the house-keeping gene is smaller than the gene of interest. Check that the two probes do not hybridise together 10 For a new probe and / or new target mRNA, the RNase conditions have to be determined empirically. The standard starting point for a fully homologous target is overnight hybridisation at 45oC with digestion at 1/5x RNase for 30 minutes at 37oC. It is easier to vary one parameter : I usually vary the RNase conditions before altering the temperature. A partly homologous target will need much less stringent conditions to allow full-length protection by the cRNA probe. Try 1/20x, 1/10x RNase at 37oC, 22oC, 16oC or even 4oC for 30 minutes or less and compare with the synthetic sense = fully homologous target mRNA. To demonstrate differences between two or more distinct although highly homologous targets, you will need very stringent RNase digestion conditions eg. 1x or 2x RNase for up to 60 minutes etc. It is easier to demonstrate differences between target mRNAs if there are two or three consecutive nucleotide differences rather than scattered base changes 11 Found that xylene cyanol (particularly at the gel purification stage) interferes with the electrophoresis of the cRNAs as they often run at the same level. Bands ugly and diffuse. 12 What can go wrong - everything! No cRNA - see section 'In vitro transcription' RNA degraded - a low molecular weight smear on gel with no large molecular weight bands. Usually operator error - not careful enough or poor reagents Multiple background bands - increase hybridisation temperature, decrease probe concentration, increase RNase digestion stringency, use PolyA+ RNA, use a different subclone for cRNA synthesis No protected bands - nothing there anyway, lost the pellet, didn't resuspend the final pellet in formamide loading buffer well enough, RNA degraded Ugly gel - acrylamide / urea degrading samples due to decomposition or they are contaminated, samples not dry before resuspending, salt left in pellets, gel run too hot >50 mA, urea left in wells, poor loading technique, gel knocked after loading and before running, wrong buffer (TAE instead of TBE), comb and spacers do not match, acrylamide in wells etc Streaking - poor resuspension in loading buffer, too large a volume used, crap in wells, wells allowed to dry out before loading etc

References

Reference #116 B. E. Faulkner-Jones, D. S. Cram, J. Kun and L. C. Harrison (1993) Localisation and quantitation of expression of two glutamate decarboxylase genes in pancreatic b-cells and other peripheral tissues of mouse and rat Endocrinol 133:2962 - 2972

Reference #6 D. A. Melton, P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn and M. R. Green (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter Nucleic Acids Res 12:7035-7056

Reference #7 P. A. Krieg and D. A. Melton (1987) In vitro RNA synthesis with SP6 RNA polymerase Methods Enzymol 155:397-415

Reference #489 D. E. Titus (1991) Promega Protocols and Applications Guide, Second Edition.

Reference #12 K. Zinn, D. DiMaio and T. Maniatis (1983) Identification of two distinct regulatory regions adjacent to the human b-interferon gene Cell 34:865-879

Reference #143 Tan Lab Library 07-94> Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular Cloning, A Laboratory Manual, Second Edition. Cold Spring Harbour Laboratory Press.

BFJ - Thesis, R. Harvey - notes from Boston including original protocol from Zinn, C. Martens, DNAX

Footnotes

1 All reagents are made up in sterile MilliQ water unless otherwise stated 1 The solution turns yellow but this does not seem to interfere with its performance 1 Use saturated formaldehyde solution - Analar or equivalent. The concentration is ~37% in water and the pH should be <4.0. If the pH is >4.0 or if there is a lot of yellow 'sludge' on the bottom of the container, get fresh stocks 1 Formamide was routinely deionised using a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation was >7.4, it was discarded 1 Make up saturated BPB solution in sterile MilliQ water 1 This is ~7.4% formaldehyde or ~2.4M. This can be reduced to ~4% / 1.2M unless the RNA of interest is particularly large 1 The gel will be ~60oC after addition of ~50 ml of reagents at room temperature 1 A paper tissue held over the mouth of the flask will catch bubbles / particle of partially dissolved agarose 1 Setting of the gel can be accelerated by placing the former in a cold room 1 This helps to free the teeth of the gel comb and prevents them tearing the gel when the comb is removed. This should always be done for low percentage and low melting point agarose gels 1 There are four common gel loading buffers which are all 6 x concentrates : I = 0.25% BPB, 0.25% XC in 40% w/v sucrose; II = 0.25% BPB, 0.25% XC in 15% w/v Ficoll 400; III = 0.25% BPB, 0.25% XC in 30% v/v glycerol and IV = 0.25% BPB in 40% w/v sucrose 1 Using too large a sample volume in the slot can result in contamination of adjacent lanes. 1 Marker RNAs may be used 1 If the RNA sample contains any alcohol, the RNA will 'creep' out of the well after loading - heat to 70oC for 10 minutes in an open tube before adding the loading buffer 1 The gel may be stained in ethidium bromide solution after electrophoresis is complete. Immerse in 0.5 mg / ml ethidium solution for ~30 - 45 minutes at room temperature 1 Formaldehyde should be difficult to smell in an adequately washed gel 1 Acrylamide is a potent, skin absorbed neurotoxin 1 Acrylamide and bis-acrylamide are slowly deaminated to acrylic acid ; the reaction is catalysed by light and alkali. Check the pH of the solution (neutral) and keep it dark and cool. Re-make solutions every few months 1 Cheaper grades of acrylamide often contain contaminants so always use sequencing grade reagents 1 Discard the solution when a precipitate forms 1 Use sequencing grade reagents. 1 Will keep for a few weeks at 4oC 1 Keep at 4oC 1 Deionise the formamide with a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation is >7.4, discard it 1 Thin gels (0.3 mm - 0.5 mm) give better resolution, do not heat up as much and are easier to fix and dry than thick (1 mm) gels. However, they are more fragile, more difficult to cast and don't allow such large amounts of sample to be loaded 1 Conventional well-forming combs can be used but, especially at low acrylamide concentrations, there are frequently problems with tearing and deformation of wells. They give good results when analysing RNase protection assays or purifying probes or nucleic acids 1 Alternatively, sharks teeth combs can be used. These give a flatter, more uniform loading-surface than the well-forming combs and reduce the risk of tearing/damage to the gel. They give good results when analysing sequencing reactions : the close proximity of the lanes allows easier reading of the sequence, but they are prone to allowing leakage of samples between wells 1 The thinner and longer the gel, the more important it is to thoroughly clean and siliconise the plates. Dirty plates will not allow even casting of the gel (air bubbles) and the gel is liable to tear when the plates are separated after electrophoresis 1 If pouring a low-percentage acrylamide gel, casting is easier if the back-plate is also siliconised 1 If a well-forming comb is to be used, do not siliconise the very top of the back-plate : the teeth of the well will collapse if the glass surface is too slippery 1 Plates washed with distilled water after previous electrophoresis just need a methanol wash and re-siliconising 1 Siliconising agents are toxic : use in a fume hood 1 This reduces the likelihood of injecting air bubbles trapped within the syringe 1 Only necessary if the full width of the gel is to be used or when desperate! 1 Gels cast the day before tend to produce smeary RNA bands - ? urea / acrylamide decomposition ? 1 When preparing low-percentage gels with well-forming combs, the wells tend to collapse. Accelerating polymerisation helps - use warmed acrylamide solutions BUT there is no leeway to make mistakes when pouring : the gel sets fast! 1 High percentage gels will polymerise well with half the amount of AP and TEMED and give more time to cast them and make mistakes 1 Allow all unused acrylamide to polymerise - essentially non-toxic - before disposal 1 Not all makes of vertical gel apparatus needs this : many have an integral metal plate positioned against the back (notched) glass plate. Also, it is usually unnecessary for small 15 x 15 cm gels and may not be a problem with a large RNase protection assay gel which will only be run ~15 cm (BPB dye front) 1 There are a variety of methods available. I use tips that are flattened front to back and slip between the two plates if a 0.4 mm spacer is used. A single tip can be used for each gel by washing it out in the anode chamber with 1 x TBE between loadings 1 Wet evenly with distilled water and blot off the excess with paper towels 1 Handle with gloves - EtBr is carcinogenic 1 This is the method to use when radiolabelled-probes are to be recovered from gels. If 32P is used as the radio-label, the gel does not have to be fixed and dried prior to autoradiography, but the resolution is poorer 1 Fixing RNase protection assay gels in methanol / acetic acid reduces the sensitivity of the assay as some of the sample is lost into the fixative 1 Wet evenly with distilled water and blot off the excess with paper towels 1 Handle with gloves - EtBr is carcinogenic 1 Radiolysis of 32P-labelled cRNA is rapid : keep for RNase protection assays for no more than 1 day after purification 1 Purified cRNA for northern hybridisations will keep a few days at -70oC 1 Formamide was routinely deionised using a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation was >7.4, it was discarded 1 Make up saturated BPB solution in sterile MilliQ water 1 An overnight immersion in 0.1% formaldehyde may be required - depends what has been done with it! 1 Formamide decomposes to ammonia - not good for RNA integrity! 1 Anecdotally, stored Proteinase K solutions adversely affect the assay 1 I have used up to 60 mg of total RNA and 200 mg of poly A+ RNA in a single 60 ml reaction 1 DNA contaminated RNA preparations are very difficult to get into solution, and will often precipitate during the hybridisation and processing steps - these will not make an aesthetic analytical gel 1 I have had to use up to 90% for very CG rich probes (eg hIGAD 65N = 80% C+G) 1 This standardises the substrate concentration for the subsequent RNase digestion step 1 The sense cRNA from the same template stock as the probe will be perfectly homologous to the antisense cRNA probe. This shows what pattern is produced between the probe and a perfectly homologous target using the chosen RNase digestion conditions. Natural RNAs encoding the same gene may have up to 3% difference at the nucleotide level between different strains 1 DNA contaminated RNA preparations are very difficult to get into solution, and will often precipitate during the hybridisation and processing steps - these will not make an aesthetic analytical gel 1 After heat denaturing the reaction prior to hybridisation, do not let the temperature fall below 45oC 1 Some probes will give better results at 50oC - trial and error 1 Increase the minimum time to 10 hours if the formamide concentration is increased to 90% 1 The accurate timing of this stage is important - limit the number of samples to ensure that they are all digested for a similar length of time 1 The timing of this stage is not as crucial as for step 12, but RNaseA will still be partially active until the sample has been phenol-extracted 1 Any residual DNA will tend to partition into the organic phase when phenol is < pH 8.0. Chloroform can be added if the interface between the two phases is not very sharp 1 When loading gel, try to keep the sample as small a volume as possible (< 7 ml) - the results from the analytical gel will look better 1 Do not use un deionised / old formamide in the loading buffer. Formamide decomposes to ammonia and urea and will degrade the RNA. Do not heat denature for too long in the loading buffer - the formamide will start to decompose

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This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95.

1 Formamide decomposes to ammonia - not good for RNA integrity! 2 Anecdotally, stored Proteinase K solutions adversely affect the assay 3 I have used up to 60 mg of total RNA and 200 mg of poly A+ RNA in a single 60 ml reaction 4 DNA contaminated RNA preparations are very difficult to get into solution, and will often precipitate during the hybridisation and processing steps - these will not make an aesthetic analytical gel 5 I have had to use up to 90% for very CG rich probes (eg hIGAD 65N = 80% C+G) 6 This standardises the substrate concentration for the subsequent RNase digestion step 7 The sense cRNA from the same template stock as the probe will be perfectly homologous to the antisense cRNA probe. This shows what pattern is produced between the probe and a perfectly homologous target using the chosen RNase digestion conditions. Natural RNAs encoding the same gene may have up to 3% difference at the nucleotide level between different strains 8 DNA contaminated RNA preparations are very difficult to get into solution, and will often precipitate during the hybridisation and processing steps - these will not make an aesthetic analytical gel 9 After heat denaturing the reaction prior to hybridisation, do not let the temperature fall below 45oC 10 Some probes will give better results at 50oC - trial and error 11 Increase the minimum time to 10 hours if the formamide concentration is increased to 90% 12 The accurate timing of this stage is important - limit the number of samples to ensure that they are all digested for a similar length of time 13 The timing of this stage is not as crucial as for step 12, but RNaseA will still be partially active until the sample has been phenol-extracted 14 Any residual DNA will tend to partition into the organic phase when phenol is < pH 8.0. Chloroform can be added if the interface between the two phases is not very sharp 15 When loading gel, try to keep the sample as small a volume as possible (< 7 ml) - the results from the analytical gel will look better 16 Do not use un deionised / old formamide in the loading buffer. Formamide decomposes to ammonia and urea and will degrade the RNA. Do not heat denature for too long in the loading buffer - the formamide will start to decompose

m7di

RNase Protection Assay - general

In this assay, uniformly labelled cRNA probes are hybridised with target mRNAs in solution. After hybridisation is complete, the reaction is incubated with RNAses specific for single-stranded RNA. The probe is thus 'protected' from RNase digestion by forming a double-stranded hybrid with the target mRNA. Digestion products are then analysed by polyacrylamide gel electrophoresis (Reference #12 K. Zinn, D. DiMaio and T. Maniatis (1983) Identification of two distinct regulatory regions adjacent to the human b-interferon gene Cell 34:865-879: Reference #7 P. A. Krieg and D. A. Melton (1987) In vitro RNA synthesis with SP6 RNA polymerase Methods Enzymol 155:397-415: Reference #6 D. A. Melton, P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn and M. R. Green (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter Nucleic Acids Res 12:7035-7056)

The high specific activity of cRNA probes and the high stability of RNA:RNA hybrids, makes RNase protection assay (RPA) up to 50 times more sensitive than conventional northern blot hybridisation (Melton, Krieg et al. 1984). Small mismatches between probe and target mRNA render the resulting hybrid vulnerable to RNase digestion, thus RPA is very specific. Also, as all target mRNA is in solution and available for hybridisation, RPAs are quantitative.

Figure of RNase protection assay - overleaf

The recombinant phagemid containing the subcloned DNA of interest is first linearised by restriction enzyme digestion (the phagemid DNA is shown in black, the RNA polymerase promoter in green, the phagemid-derived polylinker sequence lying between the RNA polymerase promoter and the subcloned DNA insert in orange and the subcloned DNA in red). In this case, the DNA insert is subcloned such that the RNA polymerase transcribes 3Õ ® 5Õ and generates cRNA complementary to the natural mRNA, or antisense cRNA. The antisense cRNA is labelled with a32P-UTP. The RNA polymerase initiation site is arrowed in green. The labelled cRNA is purified by polyacrylamide gel electrophoresis so that all the probe used for hybridisation is of unit length. Excess probe is added to the test RNA and the hybridisation is allowed to reach equilibrium.

Hybridisation of the antisense cRNA to a perfectly matched target, indicated in red, is shown on the left. The portion of the cRNA probe which is complementary to the phagemid polylinker does not form a hybrid with the target mRNA. The length of the double-stranded hybrid formed is dictated by the length of the cRNA probe, minus the polylinker segment. This in turn dictates the length of the protected species (below).

Some other mRNAs in the test sample may form imperfect hybrids with the cRNA probe because of limited regions of sequence complementarity between the two molecules. Hybridisation of the antisense cRNA to a partially homologous mRNA, indicated in blue, is shown on the right. Where the two sequences diverge, single-stranded ÔloopsÕ occur in one or other of the RNA molecules.

RNAses A and T1 digest single-stranded RNAs and double-stranded hybrids are thus ÔprotectedÕ from digestion. The segments of the cRNA probe still vulnerable to digestion after hybridisation to a perfectly homologous or to a partially homologous mRNA target are indicated by grey background shading.

The digestion products are analysed by polyacrylamide electrophoresis. The fully-protected cRNA is smaller than the undigested probe since the latter contains the additional segment of polylinker-derived RNA, and thus migrates faster. The cRNA probe annealed to the partially homologous target is digested into smaller fragments which migrate in front of the full-protected species.

Method described in (Reference #468 M. J. Elliott, B. E. Faulkner-Jones, H. Stanton, J. A. Hamilton and D. Metcalf (1992) Plasminogen activator in granulocyte-macrophage CSF transgenic mice J. Immunol. 149:3678 - 3681 and Reference #116 B. E. Faulkner-Jones, D. S. Cram, J. Kun and L. C. Harrison (1993) Localisation and quantitation of expression of two glutamate decarboxylase genes in pancreatic b-cells and other peripheral tissues of mouse and rat Endocrinol 133:2962 - 2972).

Thesis experiments Effect of radiolabelled nucleotide concentration on the proportion of full-length cRNA transcripts generated during in vitro transcription

i Methods

A single DNA template (that for insulin antisense cRNA), was used in 5 transcription reactions containing 40 mM TrisHCl (pH 8.0), 6 mM MgCl2, 2 mM spermidine HCl, 10 mM NaCl, 10 mM DTT, 100 mg/ml BSA, 20 nM linearised phagemid template DNA, 250 mM ATP, 250 mM CTP, 250 mM GTP, 1 unit ml of RNasin and 500 - 1000 units / ml of T7 RNA polymerase in a final volume of 11 ml. All reactions contained 5µl of a32P-UTP (3000 Ci / mmol, 10 mCi/ ml) at a final concentration of 1.5 mM. Unlabelled UTP was added to four of the reactions to final concentrations (labelled and unlabelled) of 3.5 mM, 11.5 mM, 21.5 mM and 26.5 mM. Reactions were incubated for 1 hour at 37oC and then diluted with 11 ml of formamide loading buffer. After 8M urea-8% polyacrylamide gel electrophoresis of the entire transcription reaction, the wet gel was exposed to Kodak XAR-5 film for 1 minute at room temperature.ii Results

Full-length cRNA transcripts of ~400 nucleotides long, were generated at all [UTP]s. However, the proportion of full-length transcripts increased and the proportion of prematurely-terminated transcripts decreased with increasing [UTP]. Maximal change was found over the range 1.5 mM - 11.5 mM, as shown.iii Comments

A pure population of unit length cRNA probes is required to detect target mRNAs by RPA. Saturating [rNTP] is >250 mM for each rNTP, with all three RNA polymerases. If the [rNTP] is lowered below 250 mM, a proportion of the resulting transcripts are prematurely terminated (Melton, Krieg et al. 1984). The proportion of these transcripts rises as the [rNTP] falls. The radiolabelled [rNTP] ranges from 1.5 mM to 60 mM per reaction, and prematurely terminated transcripts are inevitable. As the SA of the radiolabelled rNTP rises, its concentration falls. Thus raising the SA of the transcribed cRNA also increases the number of prematurely terminated transcripts. A [UTP] of 3.5 mM to 11.5 mM gave an acceptable proportion of full-length transcripts with SAs of 2.2x109 dpm/ mg and 6.8x108 dpm/µg respectively. The theoretical maximum SA using a single radiolabel is 5.1 x 109 dpm/ mg (assuming that 1 Ci = 2.22 x 1012 dpm, that 50 mCi of 3000 Ci / mmol a32P-UTP were used in each 11 ml reaction, that 100% of the radiolabel was incorporated, that a single radiolabel was used, that the average RMM of rNTPs is 330 and that 167 ng of nascent cRNA was generated). Since a proportion of transcripts are prematurely terminated at these [UTP]s, full-length cRNA was isolated by polyacrylamide gel electrophoresis and then used to assess the effect of SA on RPA sensitivity.Effect of specific activity of cRNA probes on sensitivity of RNase protection assay

i Methods

cRNA probes were synthesised at 14% and 46% of maximum SA from the same DNA template (IL-6 antisense, 295 nucleotides). Full-length transcripts were isolated from a denaturing polyacrylamide gel and then both probes hybridised with identical dilution ii Results

The higher SA probe (lanes AI and II), produced stronger autoradiographic signals at each time point than the than the lower SA probe (lanes BI and II). However, increasing the specific activity of the cRNA did not increase the sensitivity of the RPA. Both probes detected 70 pg of IL-6 sense cRNA after 1 hour at 22oC, 7 pg after 12 hours at 22oC, 0.7 pg after 12 hours at -70oC and 0.07 pg after 7 days at -70oC. The lower SA probe produced a cleaner, more aesthetic RPA, with fewer sub-bands below the full-length protected fragment.iii Comments

The SA of the lower of the two probes is comparable to that used by other workers to detect 0.1 pg of mRNA target (Melton, Krieg et al. 1984). It would be expected that the sensitivity of the RPA could be increased by using higher SA probes. Probe decay, or radiolysis, has been reported by other workers using cRNA probes (Zinn, DiMaio et al. 1983). This is clearly seen below both sets of undigested probes, but is more marked with the higher SA cRNA. The sensitivity and technical appearance of the RPA are not increased by increasing SA of the probe, presumably due to an increased rate of radiolysis of the probe.

On the basis of this result, the lower SA cRNA probes were routinely synthesised and detected between 0.05 and 0.1 pg of homologous target. No attempt was made to transcribe cRNA using two or more radiolabelled rNTPs. The choice of a32P-UTP as the radiolabelled rNTP rather than a32P-GTP, was made for the reasons given (Krieg and Melton 1987). This particular experiment was performed whilst assessing the feasibility of detecting cytokine mRNAs by RPA

Autoradiograph of denaturing polyacrylamide gel. Full-length and prematurely terminated cRNA transcripts are indicated. The final [UTP] in each reaction is given over each lane. A series of unlabelled IL-6 sense cRNA ranging from 70 pg to 0.007 pg in 10 fold dilutions. Panels A, B, C, D and E are autoradiographs of the same dried gel exposed for 30 minutes at 22oC (A), 1 hour at 22oC (B), 12 hours at 22oC (C), 12 hours at -70oC (D) and 7 days at -70oC (E). The 8 lanes in I use the higher SA probe, the 8 lanes in II, the lower SA probe. I and II are arranged as a mirror image of each other. 20 mg of tRNA was used as a negative control for both probes. Probes in lanes AI and BI, were frozen overnight at -70oC, before electrophoresis. Probes in lanes AII and BII were processed as for the test samples, but with the omission of the RNAses. Dilution series of IL-6 sense cRNA were made up after UV absorbance and EtBr-agarose gel quantitation. 70 pg of sense RNA was hybridised in lanes 1I and 1II, 7 pg in lanes 2I and 2II, 0.7 pg in lanes 3I and 3II, 0.07 pg in lanes 4I and 4II and 0.007 pg in lanes 5I and 5II

m7b

RNA dot blot and slot blot

This methods allows the rapid analysis of numerous small samples for the sequence of interest and is less time consuming than the gel electrophoresis methods. 'Dots' or 'slots' of RNA are made onto a filter using a manifold and the filter is then hybridised with a labelled probe. Partially degraded RNA can also be used with good semi-quantitative results. However, high false positive rates need careful use of appropriate controls

Protocol

Reagents

20 x SSC Standard saline citrate : 3 M NaCl, 300 mM tri-sodium citrate, pH 7.0. Add 175.3 g of NaCl and 88.2g of tri-sodium citrate to 800 ml of water. Adjust the pH to 7.0 with 1M HCl and then add water to 1 litre. Autoclave to sterilise Nitrocellulose / reinforced nitrocellulose or nylon membrane RNA loading buffer 1 x MOPS buffer (ie, 20 mM MOPS, 8 mM sodium acetate and 1 mM EDTA, pH 7.0), 7% (w/v) formaldehyde pH 4.0, 5% (v/v) sterile glycerol, 50% (v/v) deionised1 formamide and 0.025% (v/v) of saturated aqueous bromophenol blue solution 2

Equipment

BioDot or BioSlot apparatus (BioRad) Vacuum source

Methods

In advance

1 Prepare RNA samples for analysis. Have up to 20 mg in 10 ml of sterile water or 2 mm DTT with RNasin 2 Clean the dot / slot blot manifold thoroughly. Immerse in dilute detergent, rinse in running tap water and then rinse in distilled water3 3 Cut a piece of membrane to fit the dot / slot blot apparatus and pre-wet in sterile water

Method

4 Assemble the apparatus as per the manufacturers instructions - be particularly careful that no leakage is occurring between wells before loading the RNA samples - use BPB in sterile water as a marker dye 5 Rinse the wells twice with 10 x SSC and suck through the manifold under vacuum. Turn off the vacuum and add a further 50 ml of 10 x SSC to each well 6 Add two volumes of RNA loading buffer to each sample and heat denature at 70oC for 10 minutes. Chill on ice before loading 7 Load the RNA samples into the wells already containing 10 x SSC and aspirate through under vacuum 8 Rinse the wells twice with 200 ml of 10 x SSC and suck through the manifold under vacuum 9 Rinse the resulting dot blot in distilled water and briefly air-dry 10 For nitrocellulose filters - bake under vacuum for 2 hours at 80oC For nylon filters - UV cross-link the RNA in place and then bake for 15 minutes under vacuum at 80oC 11 Store the resulting dot blot at room temperature until required for hybridisation.

Notes

1 Known standards can be dotted on to the membrane to calibrate the system

2 If a manifold is not available, samples can be dotted on by hand :- care must be taken to keep the spots of uniform size, especially if semi-quantitation is required

References

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1 Formamide was routinely deionised using a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation was >7.4, it was discarded 2 Make up saturated BPB solution in sterile MilliQ water 3 An overnight immersion in 0.1% formaldehyde may be required - depends what has been done with it!

m7aiv

Recovery of RNA fragments from polyacrylamide gels

The best method is the crush and soak method. It can be sued to isolate single-stranded RNA from denaturing gels. The RNA is of high purity but the method is long and inefficient if long transcripts are to be recovered (cf. less than 30% of DNA greater than 3 kb is recovered).

Protocol

Reagents

Ethidium bromide 10 mg / ml in water Elution buffer 80% formamide in 40 mM PIPES, 1 mM EDTA and 400 mM NaCl (10 mM DTT in water if 35S-labelled RNA is being recovered) Storage buffer 10 mM DTT in sterile water 8M LiCl Isopropanol 70% ethanol

Equipment

Polyacrylamide gel electrophoresis

Methods

In advance

1 Perform polyacrylamide gel electrophoresis as described in m7aiii

Detecting the RNA of interest

Non-radiolabelled fragments

2 Take the gel-plates out of the electrophoresis chambers and lay them flat - they may be quite hot. If only the front plate has been siliconised, place the notched back plate uppermost before separating. The gel should stay attached to the front plate. Insert a metal spatula between the two plates and carefully prise apart. If the gel is sticking mainly to the back-plate (not cleaned well enough!), invert the plates and try again. The gel should remain stuck to one of the glass plates as a support 3 When the plates have been parted, lift the gel onto a piece of Whatmann 3MM paper or equivalent. Lay the glass-plate on a horizontal surface with the gel facing up and lift onto a piece of damp Whatmann paper1 2-3 cm larger than the gel. Lay the paper carefully onto the gel surface, avoiding bubbles or wrinkles. Roll these out with a glass rod. Smooth the paper onto the gel to make the two stick together. Lift one corner of the paper : the gel should stick to it and lift off the glass plate. Remove the paper and gel smoothly in a single motion and then immerse both in a shallow bath of 0.5 mg/ml ethidium bromide in 1 x TBE. Stain for 15 - 45 minutes and remove both the gel and paper2 4 Place a sheet of glad wrap over surface of a UV transilluminator and lie the gel face down on it. Peel the Whatmann paper off and transilluminate with UV light 5 Identify the band and excise it through the glad wrap 6 Crush the slice against the wall of a microfuge tube and cover with 1 - 2 volumes of elution buffer (up to 0.5 ml). Incubate with gentle agitation at 37oC. Fragments < 500 bp will elute in 3 - 4 hours, larger ones will take up to a day 7 Spin briefly to pellet the polyacrylamide then rinse the pellet with 0.5 volumes of elution buffer 8 Precipitate with one volume of isopropanol and recover the RNA by centrifugation. Even small amounts of RNA precipitate very efficiently - due to contaminating polyacrylamide ? - so there is no need to add any carrier RNA 9 Wash the RNA pellet in 70% ethanol and resuspend in 200 ml of 10 mM DTT (with RNasin if paranoid). Add 0.1 volumes of 8M LiCl and re-precipitate with an equal volume of isopropanol 10 Recover by centrifugation, wash the RNA pellet in 70% ethanol and resuspend in 10 mM DTT 11 Quantitate the RNA by UV-induced ethidium bromide fluorescence, by UV absorbance readings or by polyacrylamide gel electrophoresis on an aliquot estimated to contain 50 ng of RNA. Compare with a known standard RNA 12 Store the sample at -70oC in 10 mM DTT

Radiolabelled fragments

13 Prise the gel plates apart as in 2 above and cover the gel surface with glad wrap 14 Mark the glad wrap with a Texta pen to orientate left and right sides of the gel. Expose to X-ray film by placing the film on the bench in a dark room and the gel on its supporting plate immediately on top of it. Expose for 1 - 2 minutes in the dark and then flash the film through the glass plate. Develop the film : the markings on the glad wrap will appear translucent compared to the grey background of the film, and successfully labelled RNA will be apparent as intense black bands 15 Place the film on a transilluminating visible light source and line up the gel on its support plate using the markings on the glad wrap and the outlines of the wells. Excise the band through the glad wrap and then elute as in 5 - 12 above3,4

References

Reference #143 J. Sambrook, E. F. Fritsch and T. Maniatis (1989) Molecular Cloning, A Laboratory Manual.

Reference #151 L. G. Davis, M. D. Dibner and J. F. Battey (1986) Basic Methods in Molecular Biology.

Reference #489 D. E. Titus (1991) Promega Protocols and Applications Guide, Second Edition.

Reference #7 P. A. Krieg and D. A. Melton (1987) In vitro RNA synthesis with SP6 RNA polymerase Methods Enzymol 155:397-415

Reference #12 K. Zinn, D. DiMaio and T. Maniatis (1983) Identification of two distinct regulatory regions adjacent to the human b-interferon gene Cell 34:865-879

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1 Wet evenly with distilled water and blot off the excess with paper towels 2 Handle with gloves - EtBr is carcinogenic 3 Radiolysis of 32P-labelled cRNA is rapid : keep for RNase protection assays for no more than 1 day after purification 4 Purified cRNA for northern hybridisations will keep a few days at -70oC

m7aiii

Denaturing polyacrylamide gel electrophoresis of RNA

Protocol

Reagents

Potassium hydroxide, acetone, ethanol, methanol and Sigmacote / siliconising solution for cleaning and preparing glass gel plates. 40% acrylamide solution 38% w/v acrylamide, 2% w/v bis-acrylamide dissolved in water. Filter through a 0.2 mm filter and store in dark glass at room temperature 1,2,3 10 x TBE 890 mM Tris-borate, 20 mM EDTA, pH ~ 8.3. Add 54g of Tris base, 27.5 g of boric acid and 20 ml of 500 mM EDTA pH 8.0 to 400 ml of distilled water. Make up to 500 ml, filter through a 0.2 mm filter and store at room temperature 4 Urea 5 10% w/v ammonium persulphate solution in water6 TEMED solution7 Formamide loading buffer 80% formamide8, 1 x TBE, and 1 mg / ml bromophenol blue

Equipment

Glass plates Vertical electrophoresis tank Gel combs and spacers Metal plate.

Methods

Preparation of glass plates

1 Select a pair of glass plates of the appropriate size - long for increased resolution and wide for a large number of samples - and spacers and a gel comb. Make sure the spacers and comb are exactly the same thickness9, 10,11,12 2 With new or dirty plates, wipe the surface with KOH in methanol (5 g / 100 ml) and rinse off in distilled water until all the KOH in methanol is removed. If the plates are not too dirty, acetone alone may suffice. Handle the glass plate by the edges only and clean first with 70% ethanol, then 100% ethanol and finally 100% methanol, using a soft cloth. Allow the plates to dry before siliconising the front-plate (no rabbit-ears) with Sigmacote or any other glass-siliconising solution. Spread a few drops evenly over the surface with a soft cloth. Repeat until the friction between plate and cloth it is minimal, allowing the plate to dry in between each application13,14,15,16 3 Clean the spacers and comb with distilled water, ethanol then methanol, and check that they are all compatible when the gel-plates are assembled (but before the gel is poured!). Place the plates together with the side spacers in place and clamp the sides with bull-dog clips. Seal the bottom of the plates twice with tape, taking care with the corners : this is where most gels leak during casting

Casting the gel

4 Decide on the percentage acrylamide gel required. The figures are actually for DNA, but are a guide for RNA :-

% age acrylamide (w/v)	Effective range of	Size of RNA co-	Size of RNA co-
with BIS at 1:20	separation - bp	migrating with Xylene	migrating with
		Cyanol (33)	Bromophenol Blue
3.5	1 000 - 2 000	460	100
5.0	80 - 500	260	65
8.0	60 - 400	160	45
12.0	40 - 200	70	20
15.0	25 - 150	60	15
20.0	6 - 100	45	12

5 ~ 60 ml of acrylamide solution is required for standard sequencing gels. Make up ~ 20 ml for 15 x 15 cm gels

		20 ml				60 ml
Final %	40% stock	10%	TEMED	40% stock	10%
acrylamide	acrylamide	ammonium		acrylamide	ammonium
		persulphate			persulphate	TEMED
4%	2 ml			6 ml
5%	2.5 ml			7.5 ml
6%	3 ml			9 ml
7%	3.5 ml			10.5 ml
8%	4 ml	200 ml	20 ml	12 ml	600 ml	60 ml
9%	4.5 ml			13.5 ml
10%	5 ml			15 ml
11%	5.5 ml		10 g of		16.5 ml		30 g of
12%	6 ml		solid urea		18 ml		solid urea

Add the acrylamide and TBE to a final concentration of 1 x to the urea and make up to the final volume with MilliQ water. Mix on a rotating wheel until all the urea has dissolved 6 Add the ammonium persulphate and mix by inverting the tube 7 Add the TEMED and mix by inverting the tube 8 Draw the solution into a 20 / 60 ml syringe through a wide-bore (18 gauge) needle, then change to a narrow-bore (21 gauge) needle17. Hold the glass plates at approximately 45 degrees to the vertical, and incline to the right by balancing the plates on the bottom right-hand corner. Slowly and continuously inject the acrylamide solution down the right side of the gel, taking great care not to introduce air bubbles. Fill the bottom right-hand corner first, keeping the air-acrylamide interface smooth. Gradually alter the angle of the gel plates whilst still injecting so as to fill across the bottom of the gel. Continue injecting down one side of the gel until the acrylamide reaches the top 8 If there are any air bubbles trapped between the plates, it may be possible to remove them with a long spacer thinner than the ones used to cast the gel18 9 Lay the gel plates horizontally on support and insert the comb, taking care not to introduce bubbles around the teeth. Top up with acrylamide solution. Insert the comb so that the well 'walls' will extend to the top edge of the back (notched) glass plate. Leave to polymerise for 45 - 60 minutes at room temperature : a sharp, straight schlieren line should be visible around the teeth of the comb if polymerisation has occurred properly. The gel should be used immediately for RNA19,20,21,22

Loading and running the gel

10 Remove the comb carefully from the fully polymerised gel - it is safest to do this under buffer with the bull-dog clips still place. Immediately rinse out urea and fragments of un-polymerised acrylamide from the wells with 1 x TBE and a syringe with an 21 gauge needle 11 Remove the sealing tape and then all but the top pair of bulldog clips : take care that the two plates do not move. Clip the plates into a vertical electrophoresis apparatus (notched back-plate against the cathode chamber). 12 Clip the metal plate over the front gel plate23. The gel warms up unevenly during electrophoresis : the centre becomes hotter and the gel 'smiles'. The metal plate causes even diffusion of the heat over the gel plates and ensures even running of the samples. Fill the tank with 1 x TBE buffer and connect to the power supply (black = cathode at the top of the gel and red = anode at the bottom). Pre-electrophorese for 30 - 45 minutes at constant power (40-50 watts for a 20 cm x 40 cm long gel, roughly 1700 V) 13 Resuspend RNA samples in formamide loading buffer. Sharks-teeth combs will take up to 3 ml in volume and the wells in a 0.4 mm thick gel will usually take up to 7 ml. Heat denature the samples in loading buffer for 5 minutes at 70oC and then chill on ice 14 Turn off power to gel and rinse out the wells again with a syringe, until no urea can be seen floating into the cathode buffer. Load the samples carefully into the bottom of the well24 15 Electrophorese at constant power (40-50 watts for a 20 cm x 40 cm long gel, roughly 1700V) for the appropriate time : For a 5 - 8% sequencing-sized gel, it takes approximately 1-11/2 hours for the BPB dye front to run to the bottom (constant power, 45 - 55 watts) Using a 5% gel, to separate 400 bp from 425 bp, the BPB dye front needs to be run off the bottom twice Using a 10% gel, a 77 bp fragment is 1/3 of the way down the gel when the BPB reaches the bottom the first time

% Polyacrylamide gel	Bromophenol blue	Xylene cyanol (41)
5	35	130
6	26	106
8	19	76
10	12	55
12	8	28

The sizes are given in base pairs for RNA that co-migrates with the marker dye. RNA of the same molecular weight migrates approximately 5 - 10% slower than RNA (at 40 - 45V/cm). The differences are minimised by running the gel as fast as possible

Detecting RNA in denaturing polyacrylamide gel

Non-radiolabelled fragments

16 Take the gel-plates out of the electrophoresis chambers and lay them flat - they may be quite hot. If only the front plate has been siliconised, place the notched back plate uppermost before separating. The gel should stay attached to the front plate. Insert a metal spatula between the two plates and carefully prise apart. If the gel is sticking mainly to the back-plate (not cleaned well enough!), invert the plates and try again. The gel should remain stuck to one of the glass plates as a support 17 When the plates have been parted, lift the gel onto a piece of Whatmann 3MM paper or equivalent. Lay the glass-plate on a horizontal surface with the gel facing up and lift onto a piece of damp Whatmann paper25 2-3 cm larger than the gel. Lay the paper carefully onto the gel surface, avoiding bubbles or wrinkles. Roll these out with a glass rod. Smooth the paper onto the gel to make the two stick together. Lift one corner of the paper : the gel should stick to it and lift off the glass plate. Remove the paper and gel smoothly in a single motion and then immerse both in a shallow bath of 0.5 mg/ml ethidium bromide in 1 x TBE. Stain for 15 - 45 minutes and remove both the gel and paper26 18 Place a sheet of glad wrap over surface of a UV transilluminator and lie the gel face down on it. Peel the Whatmann paper off and photograph with UV light and / or excise the band of interest

Radiolabelled fragments - non-fixed gels

19 Prise the gel plates apart as in 16 above and cover the gel surface with glad wrap. Expose the gel to X-ray film. If probes are being made and only a short (minutes) exposure is required, invert the gel on a piece of film in the dark room. For longer exposures (a few hours), use a film-cassette 27

Radiolabelled fragments - fixed gels

20 Prise plates apart as in 16 above28 21 Lay the glass-plate on a horizontal surface with the gel facing up and lift onto a piece of damp Whatmann paper as described in 17 above and lay on a flat surface. Cover the gel in glad wrap and dry on a commercial gel dryer at 80oC for up to 2 hours 22 Remove the dried gel and peel off the glad wrap. The surface should feel smooth but not sticky 23 Autoradiograph or PhosphorImage as desired

Notes

1 RNA fragments labelled with 35S generally have to be dried for autoradiography / PhosphorImaging. A wet gel absorbs too much of the signal

2 If the paper refuses to stick to the gel, blot carefully with tissues to remove some of the water and try again

3 If the gel is torn or wrinkles badly during lifting, gentle washing with a distilled water bottle can be used to get it back into place on the paper surface. If this does not work, float the gel in a bath of distilled water and recover again onto wet Whatmann paper. NB, this gel will the take longer to dry than one which has not be immersed after electrophoresis and some of the sample will elute into the water

4 If the paper refuses to stick to the gel, blot carefully with tissues to remove some of the water and try again

References

Many but contained mainly within :- Reference #143 Tan Lab Library 07-94> Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular Cloning, A Laboratory Manual, Second Edition. Cold Spring Harbour Laboratory Press. Reference #151 Tan Lab Library 07-94> Davis LG, Dibner MD, Battey JF. 1986 Basic Methods in Molecular Biology. Elsevier. New York

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1 Acrylamide is a potent, skin absorbed neurotoxin 2 Acrylamide and bis-acrylamide are slowly deaminated to acrylic acid ; the reaction is catalysed by light and alkali. Check the pH of the solution (neutral) and keep it dark and cool. Re-make solutions every few months 3 Cheaper grades of acrylamide often contain contaminants so always use sequencing grade reagents 4 Discard the solution when a precipitate forms 5 Use sequencing grade reagents. 6 Will keep for a few weeks at 4oC 7 Keep at 4oC 8 Deionise the formamide with a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation is >7.4, discard it 9 Thin gels (0.3 mm - 0.5 mm) give better resolution, do not heat up as much and are easier to fix and dry than thick (1 mm) gels. However, they are more fragile, more difficult to cast and don't allow such large amounts of sample to be loaded 10 Conventional well-forming combs can be used but, especially at low acrylamide concentrations, there are frequently problems with tearing and deformation of wells. They give good results when analysing RNase protection assays or purifying probes or nucleic acids 11 Alternatively, sharks teeth combs can be used. These give a flatter, more uniform loading-surface than the well-forming combs and reduce the risk of tearing/damage to the gel. They give good results when analysing sequencing reactions : the close proximity of the lanes allows easier reading of the sequence, but they are prone to allowing leakage of samples between wells 12 The thinner and longer the gel, the more important it is to thoroughly clean and siliconise the plates. Dirty plates will not allow even casting of the gel (air bubbles) and the gel is liable to tear when the plates are separated after electrophoresis 13 If pouring a low-percentage acrylamide gel, casting is easier if the back-plate is also siliconised 14 If a well-forming comb is to be used, do not siliconise the very top of the back-plate : the teeth of the well will collapse if the glass surface is too slippery 15 Plates washed with distilled water after previous electrophoresis just need a methanol wash and re-siliconising 16 Siliconising agents are toxic : use in a fume hood 17 This reduces the likelihood of injecting air bubbles trapped within the syringe 18 Only necessary if the full width of the gel is to be used or when desperate! 19 Gels cast the day before tend to produce smeary RNA bands - ? urea / acrylamide decomposition ? 20 When preparing low-percentage gels with well-forming combs, the wells tend to collapse. Accelerating polymerisation helps - use warmed acrylamide solutions BUT there is no leeway to make mistakes when pouring : the gel sets fast! 21 High percentage gels will polymerise well with half the amount of AP and TEMED and give more time to cast them and make mistakes 22 Allow all unused acrylamide to polymerise - essentially non-toxic - before disposal 23 Not all makes of vertical gel apparatus needs this : many have an integral metal plate positioned against the back (notched) glass plate. Also, it is usually unnecessary for small 15 x 15 cm gels and may not be a problem with a large RNase protection assay gel which will only be run ~15 cm (BPB dye front) 24 There are a variety of methods available. I use tips that are flattened front to back and slip between the two plates if a 0.4 mm spacer is used. A single tip can be used for each gel by washing it out in the anode chamber with 1 x TBE between loadings 25 Wet evenly with distilled water and blot off the excess with paper towels 26 Handle with gloves - EtBr is carcinogenic 27 This is the method to use when radiolabelled-probes are to be recovered from gels. If 32P is used as the radio-label, the gel does not have to be fixed and dried prior to autoradiography, but the resolution is poorer 28 Fixing RNase protection assay gels in methanol / acetic acid reduces the sensitivity of the assay as some of the sample is lost into the fixative

Preparation and Examination of

Formaldehyde-Agarose Gels

Protocol

Reagents1

20 x MOPS buffer 400 mM 3-(N-morpholino)propanesulphonic acid, 160 mM sodium acetate, 20 mM EDTA, pH 7.0. Sterilise by autoclaving2 Ethidium bromide 10 mg / ml in sterile MilliQ water Agarose Formaldehyde3 RNA loading buffer 1 x MOPS buffer (ie, 20 mM MOPS, 8 mM sodium acetate and 1 mM EDTA, pH 7.0), 7% (w/v) formaldehyde pH 4.0, 5% (v/v) sterile glycerol, 50% (v/v) deionised4 formamide and 0.025% (v/v) of saturated aqueous bromophenol blue solution 5 RNA samples

Equipment

Gel former

Gel combs

Electrophoresis power supply

Methods

Preparation of agarose gel

1 Clean the gel-former and comb with distilled water, then 70% ethanol. Seal the edges of the gel-former with tape. Check that the comb sits approximately 1 mm above the gel-former when in situ 2 Make up enough buffer for both the electrophoresis tank and the gel to avoid any differences in ionic strength between them 3 For 100 ml of a 1.3% gel : melt 1.3 g of agarose in 50 ml water. Add to 20 ml of formaldehyde 6, 5 ml of 20 x MOPS and 5 ml of ethidium bromide solution in a measuring cylinder. Make the volume up to 100 ml with MilliQ water 4 Swirl to mix and then pour the gel carefully, checking for air bubbles under or between the teeth7. The final gel should be between 3 mm and 5 mm thick. Allow it to set at room temperature for 30 - 45 minutes 8, 9, 5 When fully set, pour enough buffer to cover the gel surface by ~ 1 mm and allow to stand for a couple of minutes 10. Remove the comb carefully to avoid tearing the bottom of the wells (and subsequently losing the sample), and place the gel in the electrophoresis tank. Cover to a depth of ~ 1 mm

Prepare RNA samples

6 Prepare the RNA to be analysed. The loading buffer can be diluted by up to 2 fold although generally 5 ml of aqueous RNA is added to 15 ml of RNA loading buffer11 7 Denature the RNA samples in loading buffer by heating to 70oC for 5 minutes 8 Chill on ice before loading the gel 12, 13, 14,

Electrophoresis of RNA sample

9 Load the RNA samples carefully into the slots. Connect the electrophoresis tank to a constant voltage power supply - RNA will run from black to red ie cathode to anode : make sure leads are on the right way round ! Run at 1 - 5V / cm (measured as the distance between the electrodes) until the BPB dye front has migrated the appropriate distance. Check after the BPB has run 50% of the way down the gel. The ethidium bromide will migrate the opposite way to the RNA and long electrophoresis will remove much of the ethidium from the gel15

Examination of RNA in agarose gel

10 This relies on the UV-induced fluorescence of intercalated ethidium bromide. However, formaldehyde fluoresces brightly when irradiated with UV light at 320 nm. Remove the formaldehyde by immersion in MilliQ water - up to 6 changes over up to 2 hours16 11 For a permanent record, photograph the gel when trans-illuminated by a 302 nm wavelength UV light source. This wavelength causes fluorescence of the intercalated ethidium, but reduces the amount of 'UV nicking' of the RNA. Both ethidium fluorescence and RNA nicking are maximal at 254 nm.

References

Reference #5 J. D. Allen, T. Lints, N. A. Jenkins, N. G. Copeland, A. Strasser, R. P. Harvey and J. M. Adams (1991) Novel murine homeo box gene on chromosome 1 expressed in specific haematopoietic lineages and during embryogenesis Genes Dev 5:509-520

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1 All reagents are made up in sterile MilliQ water unless otherwise stated 2 The solution turns yellow but this does not seem to interfere with its performance 3 Use saturated formaldehyde solution - Analar or equivalent. The concentration is ~37% in water and the pH should be <4.0. If the pH is >4.0 or if there is a lot of yellow 'sludge' on the bottom of the container, get fresh stocks 4 Formamide was routinely deionised using a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation was >7.4, it was discarded 5 Make up saturated BPB solution in sterile MilliQ water 6 This is ~7.4% formaldehyde or ~2.4M. This can be reduced to ~4% / 1.2M unless the RNA of interest is particularly large 7 The gel will be ~60oC after addition of ~50 ml of reagents at room temperature 8 A paper tissue held over the mouth of the flask will catch bubbles / particle of partially dissolved agarose 9 Setting of the gel can be accelerated by placing the former in a cold room 10 This helps to free the teeth of the gel comb and prevents them tearing the gel when the comb is removed. This should always be done for low percentage and low melting point agarose gels 11 There are four common gel loading buffers which are all 6 x concentrates : I = 0.25% BPB, 0.25% XC in 40% w/v sucrose; II = 0.25% BPB, 0.25% XC in 15% w/v Ficoll 400; III = 0.25% BPB, 0.25% XC in 30% v/v glycerol and IV = 0.25% BPB in 40% w/v sucrose 12 Using too large a sample volume in the slot can result in contamination of adjacent lanes. 13 Marker RNAs may be used 14 If the RNA sample contains any alcohol, the RNA will 'creep' out of the well after loading - heat to 70oC for 10 minutes in an open tube before adding the loading buffer 15 The gel may be stained in ethidium bromide solution after electrophoresis is complete. Immerse in 0.5 mg / ml ethidium solution for ~30 - 45 minutes at room temperature 16 Formaldehyde should be difficult to smell in an adequately washed gel

m7ai

Gel Electrophoresis of RNA - general

Electrophoresis through agarose or polyacrylamide gels is the standard way to separate, identify and purify nucleic acid fragments. The location of the nucleic acid within in the gel can be determined by using the fluorescent intercalating dye ethidium bromide.

Agarose gels have a smaller resolving power than polyacrylamide gels but a greater range of separation - from 200 bp to >50 kb using standard gels and electrophoresis equipment. RNAs up to 10 000 kb can be separated in agarose gels using pulsed field gel electrophoresis.

Polyacrylamide gels have enough resolving power to separate fragments differing by only one base pair in size, but their range is ~ 5 to 1000 bp. They are much more difficult to handle than agarose gels.

Formaldehyde-agarose gel electrophoresis

Agarose is a polysaccharide obtained from seaweed. There are frequently contaminants - other polysaccharides, salts and proteins - and different batches as well as different manufacturers brands vary in the level of contaminants and hence in the performance of the agarose. Agarose can be chemically modified to gel and melt at lower temperatures by the addition of hydroxyethyl groups into the polysaccharide chain.

Agarose gels are cast by completely melting the agarose in the desired buffer and then pouring into a mould to harden. RNA is negatively charged at neutral pH and when an electric field is applied, it migrates towards the anode.

RNA retains much of its secondary structure during electrophoresis unless it is first denatured. The addition of formaldehyde to the agarose gel maintains the RNA in its linear (denatured) form

The rate of migration is determined by :-

Molecular size of RNA

Linear RNA becomes orientated in an electric field in an 'end-on' position and migrates through the matrix of the gel at a rate which is inversely proportional to the log10 of the number of base pairs. Larger molecules migrate more slowly because of greater frictional drag as they try to pass through the gel matrix

Agarose concentration

Linear RNA of a given size migrates through agarose of different concentrations at different rates given by log m = logmo -KrT, where m is the electrophoretic mobility of the RNA, mo is the free electrophoretic mobility of the RNA, Kr is the retardation coefficient and T is the gel concentration

Conformation of the RNA

RNA molecules which fully or partially retain their secondary structure, migrate at different rates to fully denatured RNAs with the same molecular mass

% age of agarose (w/v) in gel	Efficient range of separation of linear RNA molecules - bp
0.3	5 000 - 60 000
0/6	1 000 - 20 000
0.7	800 - 10 000
0.9	500 - 7 000
1.2	400 - 6 000
1.5	200 - 3 000
2.0	100 - 2 000

Applied voltage.

At low voltage, the rate of migration of RNA is proportional to the voltage applied. As the voltage is increased, the mobility of larger molecules increases differentially : the effective range of separation therefore decreases with increasing voltage. For maximum resolution, run agarose gels at no more than 5V / cm (measured between the electrodes, not the gel length).

Base composition of the RNA and temperature of the gel

For agarose, neither of these parameters significantly affect the mobilities of RNA (see polyacrylamide gels)

Presence of intercalating dyes

Ethidium bromide reduces the electrophoretic mobility of linear RNA by about 15%. EtBr has greater affinity for double than single-stranded nucleic acids.

Composition of the electrophoresis buffer

In the absence of ions, RNA migrates very slowly if at all. If a 10 x buffer is used by mistake, electrical conductance is very efficient, the current generates a lot of heat and melt down happens !

Apparatus for agarose gel electrophoresis

Agarose gels are usually run as submerged horizontal slab gels. The resistance of the gel is almost the same as the buffer so a high fraction of the current passes through the gel. If a gel is to be run at high voltage, the volume of buffer should be just enough to submerge the gel and keep it wet. Apparatus used should have the following features :-

- allow easy examination of the gel by UV trans-illumination,

- should be supplied with a variety of combs to form wells of various sizes,

- should have a lid to shield electrical connections,

- allow buffer to be removed completely - important if it contains ethidium bromide and

- should allow recirculation of buffer between anode and cathode.

Polyacrylamide gel electrophoresis

Monomeric acrylamide (which is neurotoxic) is polymerised in the presence of free radicals to form polyacrylamide. The free radicles are provided by ammonium persulphate and stabilised by TEMED (N'N'N'N'-tetramethylethylene-diamine). The chains of polyacrylamide are cross-linked by the addition of methylenebisacrylamide to form a gel whose porosity is determined by the length of chains and the degree of crosslinking. The chain length is proportional to the acrylamide concentration : usually between 3.5 and 20%. Cross-linking BIS-acrylamide is usually added at a ratio of 2g BIS : 38g acrylamide.

% age acrylamide (w/v)	Effective range of	Size of RNA co-	Size of RNA co-
with BIS at 1:20	separation - bp	migrating with Xylene	migrating with
		Cyanol	Bromophenol Blue
3.5	1 000 - 2 000	460	100
5.0	80 - 500	260	65
8.0	60 - 400	160	45
12.0	40 - 200	70	20
15.0	25 - 150	60	15
20.0	6 - 100	45	12

Polyacrylamide gels are poured between two glass plates held apart by spacers of 0.4 - 1.0 mm and sealed with tape. Most of the acrylamide solution is shielded from oxygen so that inhibition of polymerisation is confined to the very top portion of the gel. The length of the gel can vary between 10 cm and 100 cm depending on the separation required. They are always run vertically with 0.5 / 1 x TBE as a buffer.

Three main advantages over agarose gels

Resolving power is such that RNA molecules differing in length by 1 base in 500 ie 0.2% can be effectively separated

They can hold up to 10 mg of RNA per slot (20 mg of RNA) without loss of resolution, a much larger amount than agarose gels

The recovered RNA or RNA is extremely pure.

Two types of polyacrylamide gel in general use

Non-denaturing gels : these are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis. The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility

Denaturing gels : these gels are polymerised with a denaturant that suppresses base pairing in nucleic acids - this is usually urea but can be formamide. Denatured RNA migrates through the gel at a rate which is almost completely independent of its composition or sequence. These gels are used for the analysis of sequencing reactions, RNase protection assays and purification of radiolabelled RNA and RNA probes

m6g

QUANTITATION OF NUCLEIC ACID CONCENTRATION

Three methods are generally used for quantitation of nucleic acids :-

i) Optical density measurements.

ii) Ethidium bromide - agarose plates.

iii) Agarose gel electrophoresis estimation with a known standard DNA

i) Optical density readings

Description

The absorbance of UV light at 260 nm wavelength by nucleic acids gives an estimate of concentration, assuming firstly that there are no protein or phenol contaminants in the solution and secondly, that the concentration of the nucleic acid is greater than 250 ng / ml. The ratio of readings taken at 260 nm and 280 nm wavelengths (both in the UV range) gives an indication of the purity of the nucleic acid.

An OD unit corresponds to the amount of nucleic acid in µg in a 1 ml volume using a 1 cm path length quartz cuvette that results in an OD260 reading of 1.

For DNA OD260 1 = 50 mm / ml,

For RNA OD260 1 = 40 mg / ml,

For single stranded oligonucleotides OD260 1 ~ 33 mg / ml

For oligonucleotides of known base sequence, the concentration of a solution can be calculated using the extinction coefficients of the bases :-

dGTP = 11.7 ml / mmole dATP = 15.4 ml / mmole

dCTP = 7.3 ml / mmole dTTP = 8.8 ml / mmole

For any given oligonucleotide, multiply the number of times each base is present by its extinction coefficient and add the resulting four numbers to get the extinction coefficient (E) of the entire molecule. The concentration is given by OD260 = E x concentration in µmoles. A more approximate estimate of concentration in µmoles is given by the total OD260 divided by 10 x the length of the oligonucleotide in bases.

The ratio of readings taken at 260 nm and 280 nm wavelengths indicates of the purity of the nucleic acid :-

For pure DNA OD260 : OD280 = 1.8

For pure RNA OD260 : OD280 = 2.0

Ratios less than these indicate contamination of the solutions with eg. protein, phenol or guanidinium and the estimates of concentration will be inaccurate. Some impurities which interfere with UV OD readings can be removed by extraction of the preparation with n-butanol.

The OD280 : OD260 ratios of the individual bases are as follows :-

dGTP = 0.66 dCTP = 0.98

dATP = 0.15 dTTP = 0.7 UTP = 0.38.

Reagents and Equipment

UV spectrophotometer.

Quartz cuvettes.

Pure sterile water.

Method

1 Blank the spectrophotometer with water (use quartz cuvettes if using ultraviolet light) Make sure the cuvette is clean, especially if estimating RNA concentration 2 Dilute a known volume of nucleic acid solution in water 3 Take readings at OD260 & OD280 4 Calculate concentration of original solution. If 5 ml of a DNA solution or 4 ml of an RNA solution are diluted in 1000 ml, the concentration in mg / ml will be 10x the OD260 reading

ii) Ethidium bromide - agarose plates

Description

This method uses the UV-induced fluorescence of ethidium bromide dye intercalated into the nucleic acid. The amount of fluorescence is proportional to the amount of nucleic acid present. Dye bound to DNA has a much stronger fluorescence in UV light than free dye. UV light at 254 nm is absorbed by the DNA and transmitted to the dye and UV at 302 nm and 366 nm is absorbed by the dye itself. The energy is re-emitted at 590 nm in the red-orange part of the visible spectrum. Very small quantities of nucleic acid can be detected this way ~ 5 ng and quantitated by comparison with a series of standards. It is also useful when the solution contains contaminants that prevent UV absorbance readings. This method is most accurate for double-stranded DNA and for RNA. The intercalation of ethidium bromide into single stranded oligonucleotides is often not strictly proportional to their mass.

Reagents and Equipment

Agarose

Sterile water

Ethidium bromide - 10 mg / ml in water

Sterile petri dish

Method

1 Melt 0.4 g of agarose in 40 ml of water by boiling briefly, cool to 60oC and add 4 ml of 10 mg / ml ethidium bromide solution 2 Pour into a petri dish and allow to harden at room temperature 3 Spot a known volume of the sample and a series of standards onto the plate - use DNA standards for DNA and RNA standards for RNA - and allow to stand for at least 30 minutes. The small molecular weight contaminants that inhibit UV absorbance readings may also enhance or quench the UV-induced fluorescence of ethidium. They will have time to diffuse away from the nucleic acid sample by allowing the plate to stand 4 Photograph the plate whilst trans-illuminated with UV light if a permanent record is required and / or if there are numerous samples to be quantitated - UV light is harmful

iii) Agarose gel electrophoresis estimation with a known standard RNA

Description

This is a variant of method ii), using UV-induced ethidium bromide-fluorescence from the test RNA and from a known amount of a RNA standard. It also allows the simultaneous assessment of the integrity of the nucleic acid. It can be used for either RNA or DNA

Reagents and Equipment

Mini-gel equipment

Agarose

Sterile water

MOPS electrophoresis buffer

Formaldehyde

Ethidium bromide - 10 mg / ml in water

Formaldehyde - formamide - glycerol - BPB loading buffer

Standard RNA This should either be serial dilutions of an RNA approximately the same size as the test RNA or eg an RNA ladder (Promega) in which the amount of RNA in each sized fragment is known and one of the fragment sizes corresponds to the test RNA. A DNA standard can be used but quantitation will be less accurate

Method

1 Make a formaldehyde-ethidium-agarose mini-gel - see m7 2 Mix a known volume of sample with RNA loading buffer 3 Mix a known quantity of standard RNA with RNA loading buffer 4 Run the gel until the BPB dye front 2/3 of the way down the gel 5 Photograph the gel on a UV transilluminator and estimate the quantity of RNA in each sample by comparing the intensity of fluorescence with the known standards.

Reference

Reference #151 Tan Lab Library 07-94> Davis LG, Dibner MD, Battey JF. 1986 Basic Methods in Molecular Biology. Elsevier. New York

FootnortesFootnotes

1 Large amounts of tissue are used, proteins etc block the interface between the two GIT-CsCl and CsCl gradients, which traps RNA and significantly reduces the yield. Also, protein and DNA pass straight through the CsCl cushion and contaminate the RNA pellet 1 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 1 If the RNA yield is expected to be high, the rotor can be stopped after 16 - 18 hours 1 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 1 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 1 If the RNA yield is expected to be high, the rotor can be stopped after 6 hours 1 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 1 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (4) and (5) reduce the amount of contamination 1 Phenol and GIT are miscible. Chloroform must be added to searate the two phases. Heating the GIT-phenol solution to 65oC increases the efficiency of the organic solvent extraction steps 1 Addition of CsCl to the GIT lysate allows a gradient to be established in the tissue lysate during ultracentrifugation. This helps prevent the interface from becoming blocked by cellular debris and increases the yield by up to 5 fold 1 If the RNA yield is expected to be high, the rotor can be stopped after 16 - 18 hours 1 High concentration CsCl precipitates out at <14oC when spun at 180 000 g 1 RNA partitions into the aquoeus phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 Usually it is possible to use 5 ml of less GIT buffer 1 If small amounts of tissue are being harvested and the appropriate homogeniser probe is available, then use an Eppendorf tube and 0.5 - 1 ml of GIT buffer 1 Very small tissue fragments can be dissolved directly in 0.5 ml of GIT at 37oC 1 RNA partitions into the aqueous phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 The pellet may be resuspended in GIT, and the phenol-chloroform extractions repeated a second or even third time. This is necessary for pancreas 1 Use 10 ml of lysis buffer per gram of tissue 1 Prolonged centrifugation makes the pellet very difficult to resuspend 1 RNA partitions into the aqueous phase and DNA partitions into the phenolic phase when the pH < 8.0. Water saturated phenol has a pH of ~ 4.0 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 The pellet may be resuspended in lithium-urea, and the phenol-chloroform extractions repeated a second or even third time. This is necessary for pancreas 1 mRNA constitutes approximately 1-5% of total RNA isolated from mammalian cells and 1g of oligo(dT) cellulose can bind up to 4 mg of poly(A+) RNA 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination 1 mRNA constitutes approximately 1-5% of total RNA isolated from mammalian cells and 1g of oligo(dT) cellulose can bind up to 4 mg of poly(A+) RNA 1 LiCl-RNA salts are insoluble in ethanol / isopropanol, whilst LiCl-DNA salts are relatively soluble. RNA from spleen and thymus particularly can become DNA contaminated and steps (5) and (6) reduce the amount of contamination

m6g

QUANTITATION OF NUCLEIC ACID CONCENTRATION

Three methods are generally used for quantitation of nucleic acids :-

i) Optical density measurements.

ii) Ethidium bromide - agarose plates.

iii) Agarose gel electrophoresis estimation with a known standard DNA

i) Optical density readings

Description

An OD unit corresponds to the amount of nucleic acid in µg in a 1 ml volume using a 1 cm path length quartz cuvette that results in an OD260 reading of 1.

For DNA OD260 1 = 50 mm / ml,

For RNA OD260 1 = 40 mg / ml,

For single stranded oligonucleotides OD260 1 ~ 33 mg / ml

For oligonucleotides of known base sequence, the concentration of a solution can be calculated using the extinction coefficients of the bases :-

dGTP = 11.7 ml / mmole dATP = 15.4 ml / mmole

dCTP = 7.3 ml / mmole dTTP = 8.8 ml / mmole

The ratio of readings taken at 260 nm and 280 nm wavelengths indicates of the purity of the nucleic acid :-

For pure DNA OD260 : OD280 = 1.8

For pure RNA OD260 : OD280 = 2.0

The OD280 : OD260 ratios of the individual bases are as follows :-

dGTP = 0.66 dCTP = 0.98

dATP = 0.15 dTTP = 0.7 UTP = 0.38.

Reagents and Equipment

UV spectrophotometer.

Quartz cuvettes.

Pure sterile water.

Method

ii) Ethidium bromide - agarose plates

Description

Reagents and Equipment

Agarose

Sterile water

Ethidium bromide - 10 mg / ml in water

Sterile petri dish

Method

iii) Agarose gel electrophoresis estimation with a known standard RNA

Description

Reagents and Equipment

Mini-gel equipment

Agarose

Sterile water

MOPS electrophoresis buffer

Formaldehyde

Ethidium bromide - 10 mg / ml in water

Formaldehyde - formamide - glycerol - BPB loading buffer

Method

Reference

Reference #151 Tan Lab Library 07-94> Davis LG, Dibner MD, Battey JF. 1986 Basic Methods in Molecular Biology. Elsevier. New York

FootnortesFootnotes

m6f

Direct isolation of poly A+ RNA

from proteinase-K-SDS-digested cell lines and tissues

This method can be used to isolate mRNA directly from cultured cells by adding the oligo (dT) cellulose to proteinase K-digested crude cell lysate. Some whole tissues can be lysed directly in the proteinase K solution, whilst others, notably pancreas, need prior extraction of total RNA using guanidinium or lithium-urea. The total RNA can then be PolyA+ selected using oligo(dT) cellulose

Protocol

General

Day 1 Homogenise tissues and isolate mRNA

Day 2 Recover mRNA, quantitate and formaldehyde-agarose gel check

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reaction

Sterile MilliQ water 10 M NaOH Proteinase K at 10 mg / ml in sterile water Oligo(dT) cellulose (Boehringer or equivalent) STE solution 100 mM NaCl, 10 mM TrisCl pH 7.4 and 1 mM EDTA pH 8.0 20% SDS in sterile water 5 M NaCl 1 M TrisCl. pH 7.4 (DEPC is unstable in Tris buffers) 500 mM EDTA pH 8.0 Binding (high salt) buffer make up just before use : the SDS will gradually precipitate out at room temperature : 500 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA pH 8.0, 0.5% SDS Wash buffer 100 mM NaCl, 10 mM TrisCl pH 7.4, 1 mM EDTA, 0.5% SDS. Elution buffer 10 mM TrisCl pH 7.4, 0.1 mM EDTA pH 8.0, 0.5% SDS Acid phenol-chloroform. 3M sodium acetate pH 6.0

Methods

In advance

1 Grow cell cultures

2 Prepare total RNA from adult organs if desired

3 Clean Polytron probe with 3 changes of distilled water.

If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA

4 Prepare the oligo(dT)cellulose

Wash 1 g of oligo(dT)cellulose in 50 ml of 400 mM sodium hydroxide in sterile water for 30 minutes at room temperature on a rotating wheel. Neutralise by washing twice in 50 ml of 500 mM TrisCl pH 7.2, followed by 6 changes of sterile water. Wash in 50 ml of binding buffer to equilibrate the salt concentration, recover by centrifugation and then resuspend in 50 ml of binding buffer

Day 1

5 Resuspend total RNA in up to 40 ml of 100 M NaCl, 10 mM TrisCl pH 7.4, 1 mM EDTA pH 8.0 and 0.5% SDS with 300 mg / ml proteinase K in a sterile 50 ml tube. Incubate for 30 - 60 minutes at 65oC 6 Homogenise cultured cells or harvested organs in up to 40 ml of 100 M NaCl, 10 mM TrisCl pH 7.4, 1 mM EDTA pH 8.0 and 0.5% SDS with 300 mg / ml proteinase K in a sterile 50 ml tube. Incubate for 30 - 60 minutes at 65oC 7 Adjust the NaCl concentration to 500 mM and add an appropriate amount of oligo(dT) cellulose in binding buffer 1 and the RNA sample allowed to bind for 2 - 4 hours at room temperature in a total volume of 50 ml on a rotating wheel 8 To remove unbound (mainly ribosomal) RNA, spin the RNA - oligo(dT)cellulose mix at 5 000 rpm for 5 minutes, decant the supernatant and resuspend in 25 ml of washing buffer warmed to 37oC. Mix on the rotating wheel for 15 minutes 9 Recover the RNA - oligo(dT)cellulose mix and wash again 25 ml of washing buffer warmed to 37oC. Mix on the rotating wheel for 15 minutes 10 Spin the RNA - oligo(dT)cellulose mix at 5 000 rpm for 5 minutes, decant the supernatant and recover bound poly(A+)RNA by eluting twice with 5 ml of 0.5% SDS in sterile water heated to 65oC. 11 Extract the eluate once with acid-phenol-chloroform. This removes any enzymatically active proteinase K and also any oligo(dT) cellulose that has been carried into the supernatant 12 Precipitate the RNA with one volume of isopropanol and 0.1 volumes of lithium chloride 2 and recover by centrifugation at 1200g for 10 minutes 13 Wash the pellet in 70% ethanol and then resuspend in 50 - 500 ml of 2 mM DTT, 1 u / ml RNasin in sterile Milli Q water 14 Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm

Day 2

15 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis 16 Store the RNA at -70oC . 17 Regenerate the oligo(dT) cellulose. Wash it well in several volumes of elution buffer, re-treat with 400 M NaOH as in 4, spin down and resuspend in 500 mM TrisCl pH 7.4. Wash for 15 minutes, spin down, check the pH of the supernatant and resuspend again in 500 mM pH 7.4 if it is still alkaline. Resuspend the neutralised oligo(dT)cellulose in sterile water, wash to remove the salt, spin down and resuspend in absolute ethanol. Store at -20oC protected from light

Notes

1 I have successfully isolated spleen, liver, macrophage and eye RNA using direct lysis in proteinase-K solution. Use large volumes (50 ml) and only a few organs 2 Brain, liver and spleen have a tendency to degrade and it is best to isolate total RNA first. Pancreas does not work at all with this method : it must first have the RNase denatured and then removed before poly(A+ ) selection. 3 Pancreas total RNA is best proteinase-K digested before oligo(dT) selection to remove any residual undenatured RNaseA. Other total RNAs can be safely resuspended directly in binding buffer without proteinase digestion. 4 The whole procedure can be performed in Econo columns. However, it is slower and the resulting mRNA has more rRNA contamination in my hands than using the 50 ml tubes 5 Recover very small amounts of PolyA+ with 20 mg of tRNA and collect in the ultracentrifuge 6 To remove any DNA, resuspend the pellet at 13 in 10 mM TrisCl pH 7.4, 1 mM EDTA, 10 mM MgCl2, and add 1 unit of RQ1 RNase-free DNaseI (Promega) per 10 ml volume. Incubate at 37oC for 30 minutes. Stop the reaction with 10 mM EDTA and 0.2% SDS. Extract once against acid-phenol, re precipitate and recover by centrifugation.

References

Reference #2 T. J. Gonda, D. K. Sheiness and J. M. Bishop (1982) Transcripts from the cellular homologs of retroviral oncogenes : distribution among chicken tissues Mol Cell Biol 2:617 - 624

Reference #462 Aviv and P. Leder (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose Proc Natl Acad Sci U S A 69:1408-12

Reference #468 M. J. Elliott, B. E. Faulkner-Jones, H. Stanton, J. A. Hamilton and D. Metcalf (1992) Plasminogen activator in granulocyte-macrophage CSF transgenic mice J. Immunol. 149:3678 - 3681

m6d

Isolation of total cellular RNA using

the lithium chloride - SDS - urea method

(Auffray-Rougeon).

Protocol

General

Day 1 Harvest and homogenise tissues. Precipitate overnight

Day 2 Isolate and purify RNA

Day 3 Formaldehyde-agarose gel check recovered RNA

Reagents All reagents must be made with sterile MilliQ water. Use

DEPC with care - it may inhibit subsequent enzyme reaction

Equipment

Polytron or equivalent

Sorval centrifuge and sterile glass (Corex) tubes for recovery of RNA

Methods

In advance

1 Clean Polytron probe with 3 changes of distilled water. If the RNA is to be used for PCR, rinse in 0.25M HCl for 15 minutes at room temperature to depurinate all contaminating DNA

Day 1

Day 2

Day 3

10 Check an aliquot of the RNA by ethidium-agarose-formaldehyde gel electrophoresis

11 Store the RNA at -70oC

Notes

References

Reference #4 C. Auffray and F. Rougeon (1980) Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumour RNA Eur J Biochem 107:303-314

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This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95.