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 that 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 Vanadyl Ribonucleosides, which are transition state analogs that 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 Guanidinium Isothiocyanate (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.
Guanidinium Isothiocyanate Isolation 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 per 30cm2 of growth area.
- 4M guanidinium isothiocyanate
- 25mM sodium citrate, pH 7.0
- 0.5% sarkosyl detergent
- 100mM 2-mercaptoethanol (use DEPC water for all stock solutions)
- To 10ml of cells in extraction solution, add the following:
- 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 the tube several times. Mix complete solution 1 minute.
- The chill solution to 4°C and centrifuge at 10,000g for 20 minutes at 4°C.
- Recover the aqueous (upper) phase and precipitate with 3 volumes of cold ethanol. Allow the solution to precipitate 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 need for further purification.
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