Preparation of Bacterial RNA

K.J. Reddy1, Michael Gilman2

1 State University of New York, Binghamton, New York, 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.4
DOI:  10.1002/0471142727.mb0404s21
Online Posting Date:  May, 2001
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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.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of High‐Quality RNA from Gram‐ Negative Bacteria
  • Basic Protocol 2: Isolation of RNA from Gram‐Positive Bacteria
  • Alternate Protocol 1: Rapid Isolation of RNA from Gram‐Negative Bacteria
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Isolation of High‐Quality RNA from Gram‐ Negative Bacteria

  Materials
  • 100‐ml E. coli culture or 500‐ml cyanobacteria culture
  • recipeStop buffer
  • recipeSTET lysing solution
  • Buffered phenol (unit 2.1)
  • Chloroform (unit 2.1)
  • 3 M sodium acetate, pH 6.0
  • 200 mM and 10 mM vanadyl‐ribonucleoside complex (VRC; GIBCO/BRL)
  • 1:1 buffered phenol/chloroform
  • DEPC‐treated water (unit 4.1)
  • Cesium chloride, solid
  • CsCl cushion: 5.7 M CsCl in 100 mM EDTA, pH 7.0
  • 100% and 70% ethanol, ice cold
  • Beckman JA‐14 and JA‐17 rotors
  • 15‐ml polypropylene tube (Sarstedt)
  • Beckman TL‐100 ultracentrifuge with TLA‐100.3 rotor and 13 × 51–mm polycarbonate centrifuge tubes, or Beckman L5‐65 ultracentrifuge with SW‐41 rotor and 14 × 89–mm ultraclear centrifuge tubes

Basic Protocol 2: Isolation of RNA from Gram‐Positive Bacteria

  Materials
  • 10‐ml bacteria culture
  • recipeLysis buffer
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 24:1 chloroform/isoamyl alcohol
  • 5 M NaCl
  • 100% and 70% ethanol, ice‐cold
  • recipeDNase digestion buffer
  • 2.5 mg/ml RNase‐free DNase I (unit 4.1)
  • TE buffer, pH 8.0 ( appendix 22)
  • Sorvall SS‐34 rotor (or equivalent)
  • Microtip sonicator

Alternate Protocol 1: Rapid Isolation of RNA from Gram‐Negative Bacteria

  Additional Materials
  • 10‐ml gram‐negative bacteria culture
  • recipeProtoplasting buffer
  • 50 mg/ml lysozyme
  • recipeGram‐negative lysing buffer
  • Diethylpyrocarbonate (DEPC)
  • Saturated NaCl: 40 g NaCl in 100 ml DEPC‐treated H 2O (stir until solution reaches saturation)
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Figures

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Literature Cited

Literature Cited
   Gilman, M.Z. and Chamberlin, M.J. 1983. Developmental and genetic regulation of Bacillus subtilis genes transcribed by sigma‐28‐RNA polymerase. Cell 35:285‐293.
   Glisin, V., Crkvenjakov, R., and Byus, C. 1974. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633‐2637.
   Hallick, R.B., Chelm, B.K., Gray, P.W., and Orozco, E.M. 1977. Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. Nucleic Acids Res. 4:3055‐3064.
   MacDonald, R.J., Swift, G.H., Przybyla, A.E., and Chirgwin, J.M. 1987. Isolation of RNA using guanidinium salts. Methods Enzymol. 152:219‐227.
   McKnight, G.S. 1978. The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in chick oviduct explant cultures. Cell 14:403‐413.
   Summers, W.C. 1970. A simple method for extraction of RNA from E. coli utilizing diethylpyrocarbonate. Anal. Biochem. 33:459‐463.
   Thomas, P.S. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. U.S.A. 77:5201‐5205.
Key Reference
   Reddy, K.J., Webb, R., and Sherman, L.A. 1990. Bacterial RNA isolation with one hour centrifugation in a table‐top ultracentrifuge. BioTechniques 8:250‐251.
  Original description of the method for isolating high‐quality RNA from E. coli and cyanobacteria.
   Summers 1970. See above.
  A brief but illuminating comparison of basic methods for isolating RNA from E. coli.
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