Unit

UNIT 4.4 Preparation of Bacterial RNA

  1. K.J. Reddy (high-quality RNA)1,
  2. Michael Gilman2

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb0404s21

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Reddy, K. and Gilman, M. 2001. Preparation of Bacterial RNA. Current Protocols in Molecular Biology. 21:I:4.4:4.4.1–4.4.7.

Author Information

  1. 1

    State University of New York, Binghamton, New York

  2. 2

    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JAN 1993

Abstract

Procedures for isolating RNA from bacteria involve disruption of the cells, followed by steps to separate the RNA from contaminating DNA and protein. Lysis strategies differ in the protocols presented in this unit, including chemical degradation of Gram-negative cell walls using sucrose/detergent or lysozyme, and sonication to break open Gram-positive cell walls. Combinations of enzymatic degradation, organic extraction, and alcohol or salt precipitation are employed in the procedures to isolate the RNA from other cellular components, and various inhibitors of ribonuclease activity (diethylpyrocarbonate, vanadyl-ribonucleoside complex, and aurintricarboxylic acid) are described. If extremely high-quality RNA is required (e.g., for gene expression studies), instructions are provided for CsCl step-gradient centrifugation to remove all traces of contaminating DNA.