Reducing the Impact of Endogenous Ribonucleases on Reverse Transcription-PCR Assay Systems

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Rapid degradation of RNA is the most important factor impeding the analysis of gene expression in human cells and tissues. Once a tissue has been removed from its normal environment, the relative rates of RNA synthesis and degradation change. This in turn gives rise to changes in the relative proportions of various RNA species within a tissue, as well as to an overall reduction of RNA concentrations. Several approaches have been used to reduce the rate at which RNA concentrations decline after tissue collection. Contamination of samples with exogenous ribonucleases (RNases), particularly those used in the laboratory during RNase treatment of DNA isolates, is universally recognized as an important cause of RNA loss. The use of meticulous laboratory technique to reduce this contamination is absolutely essential to preserve RNA substrates, but is not sufficient to permit analysis of all RNA targets. Isolation of RNA, along with the use of RNase inhibitors, such as diethyl pyrocarbonate, or chaotropic agents, such as guanidinium chloride or guanidinium isothiocyanate, often used in combination, is also helpful, but also is laborious and time-consuming. Rapid fixation of tissue samples with neutral-buffered formalin or precipitating agents, such as ethanol, destroys RNase activity, but increases the complexity …