Avoiding RNase contamination



Some guidelines on how to best avoid RNase contamination.

Common sources of contamination

 * 1) Buffers contaminated with microorganisms. (Note that the solutions need not be turbid to be problematic.)
 * 2) Pipettors. Keep a separate set of pipettors for RNA use to avoid contamination with RNases.  Avoid touching the barrel or metal ejector to the sides of tubes.

Precautions

 * 1) Keep separate a set of pipettors, glassware, plasticware, reagents, electrophoresis apparatus etc. for RNA use only.
 * 2) Store solutions in small aliquots and discard each aliquot after use.
 * 3) Clean electrophoresis apparatus.
 * 4) Wash with detergent solution.
 * 5) Rinse in H2O.
 * 6) Dry with ethanol.
 * 7) Fill with 3% solution of H2O2. (Don't use DEPC solutions because it will break down the plastic.)
 * 8) Incubate 10 mins at room temperature.
 * 9) Rinse with DEPC treated H2O.
 * 10) Handle chemicals with disposable spatulas.
 * 11) Whenever possible treat solutions with 0.1% DEPC for 1 hour at 37&deg;C and autoclave 15 mins at 15 psi.
 * 12) Remove RNases from glassware by
 * 13) Bake glassware at 300&deg;C for 4 hours or 180&deg;C or higher for several hours.
 * 14) Alternatively, soak glassware in freshly prepared 0.1% (v/v) DEPC in water or ethanol for 1 hour, drain, and autoclave (necessary to destroy any unreacted DEPC which can otherwise react with other proteins and RNA).
 * 15) Rinsing with RNaseZap followed by two rinses with RNase free H2O.
 * 16) Sterile, disposable plasticware can safely be considered RNase-free and should be used when possible.
 * 17) Metal spatulas can quickly be decontaminated by holding in a burner flame for several seconds.
 * 18) Treat plasticware with RNAseZap or DEPC.
 * 19) Use RNase-free disposable tips and tubes. Use sterile forceps to transfer items to racks.
 * 20) Use gloves from a fresh box at all times. Don't touch the gloves to any surface that might be contaminated with RNases (like yourself, door handles, refrigerators, freezers etc.).

Reference

 * 1) MolecularCloning Molecular Cloning
 * 2) AmbionDEPC RNase and DEPC Treatment (This is a very good source with systematic studies of the effect of different variables on RNase activity.
 * 3) AmbionRNaseContamination Ten Sources of RNase Contamination