Learn about the sources of RNase contamination and how you can prevent them from disrupting your experiments.
Preventing RNA degradation is trickier than preventing DNA degradation. RNases are present in all cell types from prokaryotes to eukaryotes and can sometimes survive prolonged boiling or autoclaving.
So, what are the major sources of RNases contamination in lab? Aqueous solutions and reagents…environmental exposure, as RNases are in the air, on most surfaces and in dust…and from human skin and hair.
How can you prevent this contamination?
Always wear gloves in the lab, and change them often, especially after contact with skin, hair, doorknobs, keyboards, or animals. Use RNase-free solutions, and RNase-free certified, disposable plasticware and filter tips. Maintain a separate area for RNA work and carefully clean the surfaces. Decontaminate glassware by baking at 180 degrees Celsius or higher for several hours or by soaking in freshly prepared 0.1 percent (v/v) DEPC in water or ethanol for 1 hour, followed by draining and autoclaving. Decontaminate polycarbonate or polystyrene materials (such as electrophoresis tanks) by soaking in 3 percent hydrogen peroxide for 10 minutes. Remove peroxide by extensively rinsing with RNase-free water prior to use.
How can you prevent contamination during solution preparation?
DEPC treatment of solutions is accomplished by adding 1 milliliter DEPC per liter of solution, stirring for 1 hour, and autoclaving for 1 hour to remove any remaining DEPC. However, DEPC may not always be appropriate, as it can react with amine groups, making it incompatible with Tris or Hepes buffers and reactive with certain amino acid residues.
As an alternative to DEPC, which can inhibit enzymatic reactions if not completely removed, NEB’s nuclease-free water or readily available Milli-Q purified water is sufficiently free of RNases. Solutions and buffers (for example, DTT, nucleotides, salts) should be prepared by dissolving the solid in autoclaved DEPC-treated, nuclease-free, or Milli-Q water and passed through a 0.22µm filter to sterilize.
RNase activity can also be avoided by using RNase inhibitors, such as Murine RNase Inhibitor, which requires low concentration of DTT (less than 1 millimolar) to maintain activity, making it ideal for reactions where low DTT concentration is required (like, real-time RT-PCR).
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