UNIT 4.4 Preparation of Bacterial RNA

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.