Electrophoretic analysis of RNA presents unique challenges. RNA is isolated in single stranded form, without complementary sequences. It must be fully denatured in order to obtain fractionation based on size. However, RNA molecules form complex and in some cases very stable secondary structures, which are more difficult to denature than DNA. Additionally, RNA is extremely vulnerable to degradation by RNase enzymes found either in the sample or in the processing environment. Effective procedures have been developed which allow the isolation of intact RNA, and its rigorous analysis on denaturing agarose gels. A critical component in the effectiveness of these procedures is the avoidance of RNase contamination.
RNases are small thermostable enzymes found throughout nature. In particular, RNases are found on the surface of human skin, where they are thought to play a role in defense against retroviruses. It is therefore of paramount importance that gloves be worn throughout any RNA experiment, and that any glass or plasticware possibly touched with bare hands be treated prior to use. The use of certified RNase free glassware or disposable plasticware is recommended. RNases are extremely stable enzymes. Although denatured with boiling, the enzymes renature upon cooling. Therefore, heating is not an effective method for eliminating RNase from solutions. Dry heating of glassware is effective, and glassware may be rendered RNase free by heating to 250°C for 4 hours.
Solutions which must be rendered RNase free may be treated in several ways. The most common approach is to use DEPC (Diethylpyrocarbamate) DEPC irreversibly inhibits RNase, and may then be removed by autoclaving. (NOTE: DEPC IS HIGHLY TOXIC AND VOLATILE. IT MUST BE USED ONLY IN A FUME HOOD). The limitations of DEPC are that solutions must be heated to remove the DEPC, (which would otherwise covalently modify the RNA) and that DEPC reacts with amines. Thus, to decontaminate heat labile, RNA containing or Tris buffered solutions, another method must be used. Samples containing RNA are often decontaminated during extraction by treatment with Guanidinium salts. Small volumes of Tris buffers are protected by the addition of RNase inhibitors: RNasin, a 40 kb protein or VanadylRibonucleosides, which are transition state analogs which bind to and inhibit RNase.
Preparation of RNA Samples
Isolation of intact RNA from cells depends upon the rapid inactivation of the endogenous RNase released when the cells are disrupted. The protocol presented below, based upon the system of Chomczynski and Sacchi, uses the chaotropic agent GuanidiniumIsothiocyanate (GTC) to disrupt the cells and dissolve cellular protein. RNase is inactive when dissolved in GTC in combination with reducing agents, so the disrupted cellular suspension preserves the RNA intact. The solution is then extracted with phenol to remove the RNase. This extraction is carried out at a pH of 4.5, at which DNA partitions into the organic phase, and is removed with the RNase and other proteins. Finally, the RNA is precipitated with ethanol and collected by centrifugation.
GuanidiniumIsothiocyanateIsolation of RNA
- Dissolve the tissue in 10 volumes of extraction solution. Tissue may be powdered under liquid Nitrogen, or homogenized into the buffer. Cells grown in a monolayer may be scraped into 1ml of extraction solution/30cm2 of growth area.
ExtractionSolution: / 4M GuanidiniumIsothiocyanate
25mM Sodium Citrate, pH 7.0
0.5% Sarkosyl detergent
100mM 2-Meraptoethanol
(use DEPC water for all stock solutions)
- To 10ml of cells in extraction solution, add:
1ml 2M Sodium Acetate, pH 4.5
10ml Buffer saturated phenol, pH 8
2ml chloroform: Isoamyl Alcohol, 24:1
Mix well between each addition, by inverting tube several times. Mix complete solution 1 minute.
- Chill solution to 4°C and centrifuge at 10,000g for 20 minutes at 4°C.
- Recover aqueous (upper) phase and precipitate with 3 volumes of ice-cold Ethanol. Allowsolutiontoprecipitate at -20°C for 2 hours.
- Collect RNA by centrifugation at 10,000g for 30 minutes at 4°C. Recover pellet DO NOT ALLOW PELLET TO DRY OR IT WILL NOT REDISSOLVE.
- If desired, redissolve pellet in 0.5ml of extraction solution, and repeat steps 2-5. This will give a significantly cleaner product, but with some decrease in recovery.
- Wash pellet with cold 70% Ethanol.
- Air dry pellet and redissolve in DEPC water.
- Check recovery and purity - measure A260 and A280. An A260 of 1 indicates 40µg RNA/ml in the cuvette. Pure RNA will have an absorbance ratio of A260/A280 of 2.0. 1.8 or better is generally acceptable purity for Northern Analysis or cDNA Synthesis. A ratio below 1.8 indicates a needforfurtherpurification.
Gel Electrophoresis of RNA
Agarose electrophoresis of RNA requires the inclusion of denaturing agents in the gel. In the absence of denaturants, RNA assumes compact secondary structures, which distort the relationship between molecular weight and mobility. Urea, used as a denaturant in polyacrylamide gels, disrupts the hydrogen bonds which hold the agarose gel together, and alkaline conditions, used in denaturing DNA electrophoresis in agarose, hydrolyze RNA molecules. Of the variety of denaturants which can be used for RNA analysis, all are toxic or noxious to some extent. Methylmercuric hydroxide (MMH) reacts reversibly with amino groups on RNA, and is a very effective denaturant. However, its toxicity and high volatility make its use inconvenient and hazardous. Aldehydes also react with RNA to disrupt base pairing, and are somewhat safer than MMH. Protocols are given below for using Formaldehyde or Glyoxal.
Formaldehyde Denatured RNA Gels
- Cast Gel
Dissolve 1g Agarose in 100ml of DI water. Heat to completely dissolve agarose crystals, and cool to 60°C.
Add 12 ml 10X MOPS, and 3.5 ml of 37% Formaldehyde.
0.05M Sodium Acetate
10mM EDTA
Mix well and pour gel. Insert comb and allow to set for 30-60 minutes.
- SamplePreparation:
Mix: / 4.5µl of RNA (containing 10-20µg RNA)
2µl 10X MOPS
3.5µl 37% Formaldehyde
10µl Formamide
Incubate at 55°C for 15 minutes.
Add 2µl Loading Buffer: / 50% glycerol1mM EDTA
0.25% each Bromophenol Blue and XyleneCyanol
- Runningthe Gel:
Run in 1X MOPS buffer at 10-20 V/cm for 2-3 hours, until Bromophenol Blue is 80-90% through the gel. Recirculate MOPS buffer to prevent pH drift. Use a peristaltic pump, or stop the run every 30 minutes and transfer buffer from cathode to anode and back.
GlyoxalDenatured RNA Gels
- Cast Gel
Dissolve 1.2g Agarose in 100ml of 10mM Sodium Phosphate, pH 6.9. Heat to completely dissolve agarose crystals, and cool to 60°C.
Inhibit RNases by adding sodium iodoacetate to 10mM.
Mix well and pour gel. Insertcomb and allowto set for 30-60 minutes.
- SamplePreparation:
Dissolve 10µg of RNA in 5µl of DEPC treated water. Add 6µl 6M Glyoxal, 15µl DMSO, and 3µl of 0.1M Sodium Phosphate, pH 6.9.
Note: Glyoxal must be deionized before use. After deionization, the pH of the solution should be >5.
Incubate at 50°C for 1 hour.
10mM Sodium Phosphate, pH6.9
1mM EDTA
0.25% each Bromophenol Blue and XyleneCyanol
- Runningthe Gel:
Run in 10mM Sodium Phosphate, pH 6.9 at 3-5 V/cm for 3-6 hours, until Bromophenol Blue is 60-80% through the gel. Recirculate the buffer to prevent pH drift. Use a peristaltic pump, and magnetic stir bars in the buffer chambers. If recirculation is insufficient, the pH of the buffer will rise to the point that the glyoxal will dissociate from the RNA.
Post Electrophoretic Analysis
Stain the gel by soaking in 0.05 µg/ml Ethidium Bromide. Alternately, add 4 µl of 50µl/ml Ethidium to the gel solution prior to pouring.
Visualize the gel on a UV light box. Two bands should appear in every lane. These are the ribosomal RNA bands. Determine their size based upon which species the RNA was isolated from, for they are useful molecular weight markers. The messenger RNA is visible as a smear from 10kb down to 1kb, and tRNA as a broad band near the end of the gel. Rinse the gel in 3 changes of deionized water to remove the Ethidium Bromide and formaldehyde prior to Northern Blotting.
NEXT TOPIC: Denaturing Protein Electrophoresis: SDS-PAGE
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