Question

3. Where can RNases be found? How can you prevent RNase contamination in your samples during experiments involving RNA?

Answer 1

RNases, which play important roles in nucleic acid metabolism, are found in both prokaryotes and eukaryotes, and in practically every cell type. These enzymes generally have very high specific activity, meaning tiny 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
• Environmental exposure, RNases are in the air, most surfaces and dust
• Human contact with hands and skin
• The primary source of RNases within most environments are microorganisms—namely, bacteria and fungi.

RNase contamination can be prevented during experiments involving RNA by following certain precautions as given below :

• Human body is a major source of RNase contamination. Thus, using the use of ungloved hands could easily result in RNase contamination compromising critical experiments. Use gloves and make it a point to discard used gloves and don a new pair frequently during experiments.
• Ensure that tips come from an unopened box and that tubes come from an unopened or carefully handled bag. Merely autoclaving will not destroy all RNase activity, since these enzymes are very robust and can regain partial activity upon cooling to room temperature. Always use tips and tubes that have been tested and certified RNase-free.
• If glasswares has to be used for some steps, it must be treated to remove any RNase contamination. It can be done by soaking in 0.1% Diethylpyrocarbonate (DEPC) for at least 12 hours followed by thorough washing in DEPC-treated water and autoclaving.