chapter  2
25 Pages

Isolation and Purification of RNA

Contents Ribonucleic acid (RNA) extraction and handling are technically more challenging than deoxyribo-nucleic acid (DNA) manipulation due to the ubiquity and stability of RNases and to the presence of a reactive 2′-OH on RNA ribose residues.1,2 Consequently, significant measures are taken to 0-8493-0815-1/04/$0.00+$ 1.50

preserve RNA integrity throughout purification. Rapid inactivation of RNases released during cell lysis is critical for the isolation of undegraded RNA. All RNA extraction buffers, therefore, contain powerful inactivating denaturants such as guanidinium salts or detergents. Since airborne bacteria and the skin (e.g., hands) are also sources of RNases, all solutions and labware used in RNA isolation should be treated to remove RNases, and gloves must be worn at all times. Successful RNA extraction relies on good laboratory practice and RNase-free technique. We recommend the following precautions for RNA work:

1. All glassware and reuseable plasticware used in solution preparation, storage, and RNA extraction should be treated to remove residual RNase activity and be autoclaved before use. Most protocols call for treatment of labware with 0.1% DEPC (diethylpyrocarbonate) in distilled, deionized H2O (ddH2O) (stir to mix) for several hours to overnight, followed by several rinses with autoclaved ddH2O and autoclaving. However, DEPC is a suspected carcinogen and should be handled carefully and used only in a fume hood. Alternatively, we have found AbSolve™ (Cat. #NEF971, NEN Life Science Products, Boston, MA), a liquid-concentrate cleaner formulated to destroy RNases, to be equally effective and more user-friendly. To use, immerse all labware in 2% AbSolve in ddH2O for several hours to overnight and rinse with autoclaved ddH2O prior to autoclaving.