RNase contamination, prevention,

All solutions used for reverse transcription must be free of RNase. If all solutions are prepared with RNase-free water in disposable plastics and no contact with RNase-contaminated spatula or pH probes has occurred, the solutions need not be treated any further. Otherwise, to decontaminate the solutions add diethyl pyrocarbonate (DEPC) (e.g., Sigma, St. Louis, MO) to the solution to a final concentration of 0.1%, shake, let sit overnight with loosened caps, and then autoclave for 15 min. Note that DEPC might be carcinogenic and that solutions containing Tris cannot be decontaminated with DEPC, but must be prepared with special caution to prevent any RNase contamination. 

Compared with DNA, more care has to be taken when working with RNA to prevent any RNase contamination. Use of gloves (hands are the major source of RNase), diethyl pyrocarbonate (DEPC)-treated solutions, disposable tubes, pipet tips from freshly opened bags, and RNasin (a potent ribonuclease inhibitor) in the labeling reaction, minimizes the risk of RNA degradation. 

HIV, as already mentioned, is a retrovirus, i.e., the starting material submitted to amplification is RNA. This genetic material is very labile and can be efficiently hydrolyzed by RNases, which, on the contrary, are very stable and ubiquitous enzymes. Thus, special care must be taken to prevent contamination of the extraction area and equipment by these enzymes. This topic will be addressed in Subheading 1.3. 

For the mRNA extraction experiment, one of the most important points is to prevent contamination, such as RNase or RNA molecules. To overcome this problem, careful washing of the instruments is critically important during the preparation of the experiment. The procedures are as follows. 

Successful RNA preparation depends on the inhibition of both endogenous RNase activity liberated on cell lysis and contamination of preparations by exogenous RNases. In this method treatment of equipment and solutions with diethyl pyrocarbonate (DEPC), a strong inhibitor of RNases, is used to prevent degradation of samples. In addition working quickly and keeping preparations on ice whenever possible will help to minimize problems with RNase activity. 

In contrast to radioactively labeled DNA probes, the medium for prehybridization and hybridization is adapted to RNA (2) mainly to prevent contamination by RNases. However, diethyl pyrocarbonate (DEPC) treatment of the water is not necessary. Good quality distilled water can be used instead to prepare the solutions as some contain SDS, which inhibits RNase activity (washing solutions) whereas others are kept as highly concentrated stock solutions (20X SSC), are autoclaved and stored at 4°C (TN buffer, alkaline TNM buffer, and TE buffer), or are made freshly (SSC dilutions, blocking buffer, ABC solution, and color solution). 

Enzymes should be handled with care to avoid contamination. Use a fresh pipette (or tip) for each aliquot that is removed from the parental vial. Never return unused material to the parental vial. Wear gloves to prevent contaminating the enzyme with proteases, DNases, RNases, and inhibitors often found on fingertips. Never pipette by mouth. 

---