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). RNases are a ubiquitous family of enzymes, present in virtually all living cells, which 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, including a checklist, which each person in the lab is obligated to follow. The methodologies for RNase control are closely tied to the protocols used to isolate RNA, including the formulation of the lysis buffer.