Avoiding Ribonuclease Contamination
RNA is more susceptible to degradation than DNA, due to the ability of the 2´ hydroxyl groups adjacent to the phosphodiester linkages in RNA to act as intramolecular nucleophiles in both base- and enzyme-catalyzed hydrolysis. Whereas deoxyribonucleases (DNases) require metal ions for activity and can therefore be inactivated with chelating agents (e.g. EDTA), many ribonucleases (RNases) bypass the need for metal ions by taking advantage of the 2´ hydroxyl group as a reactive species.
Many microbial ribonucleases, including E. coli RNase I and RNase M (1), apparently share many properties with bovine pancreatic RNase A. RNase A is a single-strand specific endoribonuclease that is resistant to metal chelating agents and can survive prolonged boiling or autoclaving. RNase A-type enzymes rely on active site histidine residues for catalytic activity (2) and can be inactivated by the histidine-specific alkylating agent diethyl pyrocarbonate (DEPC).
Sources of RNase contamination:
It is important to realize that, even though New England Biolabs' enzymes certified for RNA work have been purified free of ribonucleases, it is possible to reintroduce RNases during the course of experimentation via aqueous solutions or from the environment at large. Autoclaving a solution will kill contaminating bacteria, but RNases liberated from the dead bacteria will still be active. Additionally, ungloved fingers can introduce bacteria into solutions resulting in RNase contamination. RNase contamination can be avoided by using RNase-free solutions and following a few common sense laboratory procedures.
Laboratory precautions (3,4):
Always wear gloves when working with RNA. It is a good idea to maintain a separate area for RNA work that has its own set of pipettors. This is especially important if your work requires the use of RNase A (e.g. plasmid preps). Sterile, disposable plasticware can safely be considered RNase-free and should be used when possible. Metal spatulas can quickly be decontaminated by holding in a burner flame for several seconds. Any piece of glassware that was ever in common use should be viewed with suspicion. Contaminating RNases can be inactivated by baking glassware at 180°C or higher for several hours. Alternatively, glassware can be soaked in freshly prepared 0.1% (v/v) DEPC in water or ethanol for 1 hour, drained, and autoclaved (necessary to destroy any unreacted DEPC which can otherwise react with other proteins and RNA). DEPC will destroy polycarbonate or polystyrene materials (e.g. electrophoresis tanks), which should instead be decontaminated by soaking in 3% hydrogen peroxide for 10 minutes. Remove peroxide by extensively rinsing with RNase-free water (see below) prior to use.
Preparation of solutions (3,4):
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 hydrolyze any remaining DEPC. Compounds with primary amine groups (e.g. Tris) will react with DEPC. Consequently, Tris buffers should be prepared by dissolving Tris base (from a fresh bottle reserved for RNA work) in DEPC-treated and autoclaved water, adjusting the pH (with an electrode reserved for RNA work), and re-autoclaving to sterilize. Solutions of thermolabile materials (e.g. DTT, nucleotides, manganese salts) should be prepared by dissolving the solid (highest available purity) in DEPC-treated and autoclaved water and passing the solution through a 0.2 µm filter to sterilize. As an alternative to DEPC, which can inhibit enzymatic reactions if not completely hydrolyzed, we have found that Milli-Q™ (Millipore) purified water is sufficiently free of RNases for most RNA work.
Inhibitors of ribonucleases:
RNA can also be protected from RNase activity by using an RNase inhibitor. Ribonucleoside vanadyl complex (NEB #S1402S) is a transition-state analog inhibitor of RNase A-type enzymes, with Ki = 1 X 10-5 M. Unfortunately, the complex also inhibits many other enzymes used in RNA work. One exception is reverse transcriptase, so this complex has proved useful in protecting cellular mRNA from degradation during purification prior to cDNA synthesis (5). An angiogenin-binding protein isolated from human placenta is a better RNase inhibitor (Ki = 4 x 10-14 M; 6) and is specific for RNases, but it is much more expensive. Consequently, this inhibitor is useful only for small-scale applications (e.g. in vitro transcription).
References:
1. Meador III, J. and Kennell, D. (1990) Gene 95, 1-7.
2. Fersht, A.R. (1977) Enzyme Structure and Mechanism Freeman, Reading, PA, 325-329.
3. Blumberg, D.D. (1987) Methods Enzymol. 152, 20-24.
4. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press, Cold Spring Harbor, NY, 7.3-7.5.
5. Berger, S.L. (1987) Methods Enzymol. 152, 227-234.
6. Lee, F.S. and Vallee, B.L. (1990) Proc. Natl. Acad. Sci. USA 87, 1879-1883.
