Avoiding Ribonuclease Contamination, NEB

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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).

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Sources of RNase contamination:

New England Biolabs' enzymes certified for RNA work have been purified free of ribonucleases. However, it is possible to reintroduce RNases during the course of experimentation through various sources: Aqueous solutions Note: Autoclaving will kill contaminating bacteria, but RNases liberated from the dead bacteria will still be active. Environmental exposure Human contact

Laboratory precautions (3,4):

RNase contamination can be prevented by following a few common sense laboratory procedures: Use RNase-free solutions. Always wear gloves. 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). Use sterile, disposable plasticware whenever possible. Decontaminate metal spatulas by holding in a burner flame for several seconds. Avoid common use glassware. Decontaminate glassware by baking at 180C 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 (see below) prior to use.

Preparation of solutions (3,4):

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 hydrolyzed, 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 hydrolyze any remaining DEPC. Note: compounds with primary amine groups (e.g., Tris) will react with DEPC. Tris buffers should be prepared by dissolving Tris base (from a fresh bottle reserved for RNA work) in autoclaved DEPC-treated or Milli-Q 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 autoclaved DEPC-treated or Milli-Q water and passing the solution through a 0.2 m filter to sterilize.

Inhibitors of ribonucleases:

RNA can also be protected from RNase activity by using one of the following RNase inhibitors: Ribonucleoside Vanadyl Complex (NEB #S1402) is a transition-state analog inhibitor of RNase A-type enzymes, with K = 1 X 10
i -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

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Avoiding Ribonuclease Contamination, NEB

http://www.neb.com/nebecomm/tech_reference/general_data/avoiding_c...

from degradation during purification prior to cDNA synthesis (5). RNase Inhibitor, (NEB #M0307), an angiogenin-binding protein isolated from human placenta, is a better inhibitor (K = 4 x 10
i -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. 2. 3. 4. Meador III, J. and Kennell, D. (1990) Gene, 95, 17. Fersht, A.R. (1977) Enzyme Structure and Mechanism Freeman, Reading, PA, 325329. Blumberg, D.D. (1987) Methods Enzymol., 152, 2024. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd ed.), (pp. 7.37.5). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 5. Berger, S.L. (1987) Methods Enzymol., 152, 227234. 6. Lee, F.S. and Vallee, B.L. (1990) Proc. Natl. Acad. Sci. USA, 87, 18791883.

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