It is important to remember that RNA will be degraded only in the presence of ribonuclease. Ribonuclease may be present in the lab from previous experiments (e.g. RNAse protection assays), from glassware that once contained serum or from human skin. The likelihood of ribonuclease contamination may be alleviated by performing experiments with RNase in dedicated spaces or in separate laboratories, by using disposable plasticware rather than reusable glassware and by always wearing clean gloves when working with RNA. The two most important aspects of this procedure are to have a visible RNA pellet after the initial precipitation step and accurate pipetting. Due to the sensitivity of the PCR, it is possible to obtain PCR signals by amplifying cDNA synthesized from <1 ng of total RNA. However, we have observed the most consistent results when an RNA pellet is visible following the precipitation. Accurate and conscientious pipetting is critical to reducing error and preventing cross contamination.
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