RNA is more susceptible to degradation than DNA, due to the ability of the 2´ hydroxyl groups to act as nucleophiles. Many ribonucleases (RNases) bypass the need for metal ions by taking advantage of the 2´ hydroxyl group as a reactive species. These RNases are resistant to metal chelating agents and some of them, like RNase A family enzymes, can survive prolonged boiling or autoclaving. RNase A-type enzymes rely on active site histidine residues for catalytic activity (1) and can be inactivated by the histidine-specific alkylating agent diethyl pyrocarbonate (DEPC).
Sources of RNase Contamination
RNases are found in all cell types and organisms from prokaryotes to eukaryotes. These enzymes generally have very high specific activity, meaning miniscule amounts of contamination in an RNA sample is sufficient to destroy the RNA. The major sources of RNase contamination in a typical laboratory include:
- Aqueous solutions, reagents used in experiments
- Exposure to RNase from environmental sources (lab surfaces, aerosols from pipetting, ungloved hands, etc.)
- Contaminated reagents
Laboratory Precautions (2,3)
New England Biolabs' enzymes have been purified free of ribonucleases. However, it is possible to reintroduce RNases during the course of experimentation through various sources. RNase contamination can be prevented by following a few common sense laboratory procedures:
- Always wear gloves during an experiment and change them often, especially after contact with skin, hair or other potentially RNase-contaminated surfaces such as doorknobs, keyboards and animals.
- Use RNase-free solutions. Use RNase-free certified, disposable plasticware and filter tips whenever possible.
- Maintain a separate area for RNA work. Carefully clean the surfaces.
- Decontaminate glassware by baking at 180°C or higher for several hours or by soaking in freshly prepared 0.1% (v/v) DEPC in water or ethanol for 1 hour, followed by draining and autoclaving. Autoclaving will destroy any unreacted DEPC which can otherwise react with other proteins and RNA.
- Decontaminate polycarbonate or polystyrene materials (e.g. electrophoresis tanks) by soaking in 3% hydrogen peroxide for 10 minutes. Remove peroxide by extensively rinsing with RNase-free water prior to use.
Preparation of Solutions (2,3)
Preparation of solutions using the following suggestions can help prevent RNase contamination:
- As an alternative to the historic use of DEPC, which can inhibit enzymatic reactions if not completely removed, we have found that Milli-Q™ (Millipore) purified water is sufficiently free of RNases for most RNA work.
- DEPC treatment of solutions is accomplished by adding 1 ml DEPC (Sigma) per liter of solution, stirring for 1 hour, and autoclaving for one hour to remove any remaining DEPC. [Note: Compounds with primary amine groups (e.g., Tris) which will react with DEPC, cannot be DEPC-treated. Other compounds, which are not stable during autoclaving, cannot be DEPC-treated].
- Solutions and buffers (e.g. DTT, nucleotides, manganese salts) should be prepared by dissolving the solid (highest available purity) in autoclaved DEPC-treated or Milli-Q water and passing the solution through a 0.22 µm filter to sterilize.
Inhibitors of Ribonuclease
RNA can also be protected from RNase activity by using one of the following RNase inhibitors:
- RNase Inhibitor, Murine, (NEB #M0314) is the Murine version of RNase inhibitor. It has the same inhibition profile of human or porcine inhibitors but is more stable due to improved resistance to oxidation (4). The inhibitor requires low concentration of DTT (< 1 mM) to maintain activity, making it ideal for reactions where low DTT concentration is required (e.g., real-time RT-PCR).
- RNase Inhibitor, Human Placenta, (NEB #M0307), a recombinant protein of human placenta, is a specific inhibitor for RNases A, B and C. Similar to the Murine inhibitor, it is compatible with many enzymatic reactions involving RNA (e.g., in vitro transcription, RT-PCR, ligation, etc.).
- Ribonucleoside Vanadyl Complex (NEB #S1402) is a transition-state analog inhibitor of RNase A-type enzymes with Ki = 1 X 10-5 M. This complex is compatible with many RNA isolation procedures, but it should not be used in the presence of EDTA. The complex also inhibits many other enzymes used in RNA work (5).
- Fersht, A.R. (1977) Enzyme Structure and Mechanism Freeman, Reading, PA, 325-329.
- Blumberg, D.D. (1987) Methods Enzymol., 152, 20-24. PMID: 2443792
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd ed.), (pp.7.3-7.5). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
- Kim, B.M. et al. (1999) Protein Science, 8, 430-434. PMID: 10048337
- Berger, S.L. (1987) Methods Enzymol., 152, 227-234. PMID: 2443795
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