Preparation of Cytoplasmic RNA from Tissue Culture Cells

Michael Gilman1

1 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.1
DOI:  10.1002/0471142727.mb0401s58
Online Posting Date:  May, 2002
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Abstract

The protocol described in this unit is a fast and streamlined procedure for preparing total cytoplasmic RNA from many cultures simultaneously for nuclease protection analysis. It is scaled for small cultures‐‐1 to 2 dishes of adherent cells or 10 to 20 ml of a suspension culture. The procedure works well for many cell types. If full‐length RNA is required, ribonuclease inhibitors should be added to the lysis buffer (as described in this unit) or the guanidinium isothiocyanate method should be used. Finally, if RNA is isolated from transiently transfected cells, a is provided for the treatment of RNA with deoxyribonuclease to remove transfected DNA. This modification is especially critical if the RNA is to be assayed by nuclease protection using uniformly labeled probes.

     
 
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Table of Contents

  • Section I: Preparation of RNA from Eukaryotic and Prokaryotic Cells
  • Basic Protocol 1: Preparation of Cytoplasmic RNA from Tissue Culture Cells
  • Support Protocol 1: Removal of Contaminating DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Cytoplasmic RNA from Tissue Culture Cells

  Materials
  • Cell monolayer or suspension
  • Phosphate‐buffered saline, ice cold (PBS; appendix 22)
  • recipeLysis buffer, ice cold (see recipe)
  • 20% (w/v) sodium dodecyl sulfate (SDS)
  • 20 mg/ml proteinase K
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 24:1 chloroform/isoamyl alcohol
  • 3 M recipeDEPC‐treated (see recipe) sodium acetate, pH 5.2 ( appendix 22)
  • 100% ethanol
  • 75% ethanol/25% 0.1 M recipeDEPC‐treated (see recipe) sodium acetate, pH 5.2
  • Beckman JS‐4.2 rotor or equivalent
  • Rubber policeman
  • Additional reagents and equipment for removing contaminating DNA (see protocol 2)
CAUTION: Diethylpyrocarbonate (DEPC) is a suspected carcinogen and should be handled carefully ( appendix 1H).IMPORTANT NOTE: Water and sodium acetate should be treated with DEPC (see recipe).

Support Protocol 1: Removal of Contaminating DNA

  • TE buffer, pH 7.4 ( appendix 22)
  • 100 mM MgCl 2/10 mM dithiothreitol (DTT; see appendix 22 for both components)
  • recipe2.5 mg/ml RNase‐free DNase I (see recipe)
  • 25 to 50 U/µl placental ribonuclease inhibitor (e.g., RNAsin from Promega Biotec) or vanadyl‐ribonucleoside complex
  • recipeDNase stop mix (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Berk, A. J. and Sharp, P. A. 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease‐digested hybrids. Cell 12:721‐732.
   Favoloro, J., Treisman, R., and Kamen, R. 1980. Transcription maps of polyoma virus‐specific RNA: Analysis by two‐dimensional nuclease S1 gel mapping. Meth. Enzymol. 65:718‐749.
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