Decontamination and inhibition of ribonucleases

Decontamination and inhibition of ribonucleases

It is common knowledge in most laboratories that RNA is more susceptible to degradation than DNA. Unlike Deoxyribonucleases (DNases), which require metal ions for activity (and can therefore easily be inactivated with chelating agents such as EDTA), ribonucleases (RNases) have virtually no cofactor requirements and advantage of the 2’ hydroxyl groups adjacent to the phosphodiester linkages in RNA as a reactive species.

Sources for RNase contamination in the Laboratory
It is possible to contaminate samples with RNases during the course of an experiment in various ways:

1. Autoclaving buffers and solutions will not inactivate RNases and may actually introduce them into buffers e.g. by liberating them from contaminating bacteria.
2. Ungloved hands represent a source for introducing bacteria into solutions resulting in RNase contamination and can even contaminate solutions directly with released cellular RNases.
3. Airborne contaminants settling on the surface of a solution of buffer could also carry RNases.

Laboratory precautions
Microbial ribonucleases share many properties with bovine pancreatic RNase A. The pancreatic ribonucleases are small enzymes (15 kd) and consist of a single polypeptide chain with four disulfide bridges. RNase A is a single-strand specific endoribonuclease that is active over a wide pH range, is resistant to metal chelating agents and can survive prolonged boiling or autoclaving ⁽¹⁾ . RNase A-type enzymes rely on histidine residues within the active site for catalytic activity and can be inactivated by the alkylating agent diethyl pyrocarbonate (DEPC) which modifies these residues. RNase contamination can effectively be avoided by using RNase-free solutions such as Eppendorf Molecular biology reagents and following a few common sense laboratory procedures:

• Always wear gloves when working with RNA.
• Maintain a separate area for RNA work that has an own set of pipettes, pipette tips, Eppendorf tubes, buffers, and reagents. This is especially important if your work requires the use of RNases for e.g. plasmid preparations.
• Sterile disposable plasticware produced under clean room conditions is RNase-free and should be used when possible. Eppendorf Biopur tips and tubes provide this quality feature in addition to being DNase free.
• Metal tools like spatulas can quickly be decontaminated by holding in a burner flame for several seconds. Contaminating RNases on glassware can be inactivated by baking the glassware at 180°C or higher for several hours.

Alternatively, glassware can be soaked in freshly prepared 0.1% 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 the adenine residues of RNA). As DEPC will attack polycarbonate (e.g. centrifuge tubes) or polystyrene (e.g. standard microtiter plates) decontamination of these materials can be achieved by soaking in 3% hydrogen peroxide for 10 minutes. Residuals of pero xide can be removed by extensively rinsing with RNase-free water^{1., 2 .} . An alternative protocol uses a 1 N NaOH soak of 1 hour at 37°C. After soaking in NaOH the labware is then washed extensively in RNase-free water.

RNase decontamination of buffers and solutions
DEPC treatment of solutions is accomplished by adding 1 ml DEPC per liter of solution, stirring for 1 hour, and autoclaving for one hour to hydrolyze any remaining DEPC into CO₂ and Ethanol. Compounds with primary amine groups (e.g. Tris) will react with DEPC. Consequently, Tris buffers should be prepared by dissolving Tris base (from 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^{2., 3.} . As an alternative to DEPC, which inhibits enzymatic reactions and modifies both RNA and Tris if not completely hydrolyzed, we recommend the use of RNase-free Molecular Biology Grade Water.

Use of ribonucleases inhibitors
A very convenient and effective way to protect RNA from decomposing RNase activity is the use of an RNase inhibitor. Eppendorf Prime RNase Inhibitor is a protein of non-human origin that binds non-covalently to RNase and inhibits the same type of ribonucleases as human placental RNase inhibitors (HPRI), including RNases A, B, and C. Prime RNase Inhibitor is stable under a broad range of pH, DTT concentrations and temperatures. It inhibits greater than 90% of RNase activity in reactions where an equal amount of HPRI inhibits only 50%.

References:
1. Blackburn, P., and Moore S. (1982) The Enzymes. Academic Press, NY, 317.
2. 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.
3. Blumberg, D.D. (1987) Methods Enzymol. 152, 20�24.