Chapter 2 - Creating a Ribonuclease-Free Environment

Abstract

This chapter focuses on the process of isolation of ribonucleic acid (RNA) from biological sources enriched in ribonucleases (RNase) and then protecting the RNA from degradation in the ensuing protocols. RNases are a ubiquitous family of enzymes, present in virtually all living cells, that can degrade RNA molecules through both endonucleolytic and exonucleolytic activity. They are remarkably stable enzymes and their control is of paramount importance. It is incumbent upon the investigator to ensure that both equipment and reagents are purged of nucleases from the onset of the experiment. The most straightforward procedure to this end is for the lab to develop a detailed standard operating procedure (SOP), including a checklist, which each person in the lab is obligated to follow. Protocols for the isolation of RNA begin with cellular lysis mediated by buffers that typically fall into one of two categories depending on the required degree of cellular disruption and concomitant RNase inhibition: (1) those consisting of harsh chaotropic agents such as one of the guanidinium salts, which disrupt the plasma membrane and subcellular organelles and which simultaneously inactivate RNase; and (2) those that gently solubilize the plasma membrane while maintaining nuclear and other organelle integrity, such as the nonionic, hypotonic lysis buffers. The disadvantage of using gentle lysis buffers is that the lysis buffer alone is not sufficiently chaotropic to fully inhibit RNase activity; and the drawback of harsh chaotropic procedures is that the investigator is not able to discriminate between RNAs from various subcellular compartments once various subpopulations have been mixed. There are many commercial products available for RNase control. Failure to do so will inevitably result in a useless sample of degraded RNA and marked reduction in lab productivity.