Ribonuclease Protection Assay

Michael Gilman1

1 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.7
DOI:  10.1002/0471142727.mb0407s24
Online Posting Date:  May, 2001
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Abstract

Sequence‐specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe.

     
 
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Table of Contents

  • Support Protocol 1: Gel Purification of RNA Probes
  • Support Protocol 2: Preparation of Template DNA
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Diethylpyrocarbonate (DEPC)
  • recipe5× transcription buffer
  • 200 mM dithiothreitol (DTT)
  • recipe3NTP mix (ATP, UTP, and GTP at 4 mM each; unit 3.4)
  • recipe[α32P]CTP (10 mCi/ml, 400 to 800 Ci/mmol)
  • Placental ribonuclease inhibitor (e.g., RNAsin from Promega Biotec)
  • protocol 30.5 mg/ml template DNA ( protocol 3)
  • recipeBacteriophage RNA polymerase (unit 3.8)
  • 2.5 mg/ml RNase‐free DNase I (unit 4.1)
  • 10 mg/ml tRNA (unit 4.6)
  • 25:24:1 phenol/chloroform/isoamyl alcohol
  • 2.5 M ammonium acetate
  • 100% ethanol
  • 75% ethanol/25% 0.1 M sodium acetate, pH 5.2
  • recipeHybridization buffer
  • recipeRibonuclease digestion buffer
  • 40 µg/ml ribonuclease A
  • 2 µg/ml ribonuclease T1
  • 20% (w/v) sodium dodecyl sulfate (SDS)
  • 20 mg/ml proteinase K (store at −20°C)
  • recipeRNA loading buffer
  • Additional reagents and equipment for phenol extraction (unit 2.1) and denaturing polyacrylamide gel electrophoresis (unit 2.12 & unit 7.6)

Support Protocol 1: Gel Purification of RNA Probes

  Additional Materials
  • TBE buffer ( appendix 22)
  • recipeElution buffer
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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.
   Butler, E.T. and Chamberlin, M.J. 1982. Bacteriophage SP6‐specific RNA polymerase. I. Isolation and characterization of the enzyme. J. Biol. Chem. 257:5772‐5778.
   Chamberlin, M. and Ryan, T. 1982. Bacteriophage DNA‐dependent RNA polymerase. In The Enzymes, Vol. XV (P. Boyer, ed.), pp. 87‐108. Academic Press, NY.
   Kassevetis, G.A., Butler, E.T., Roulland, D., and Chamberlin, M.J. 1982. Bacteriophage SP6‐specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription. J. Biol. Chem. 257:5779‐5787.
   Ley, T.J., Anagnou, N.P., Pepe, G., and Nienhuis, A.W. 1982. RNA processing errors in patients with β‐thalassemia. Proc. Natl. Acad. Sci. U.S.A. 79:4775‐4779.
   Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 12:7035‐7056.
   Weaver, R.F. and Weissman, C. 1979. Mapping of RNA by modification of the Berk‐Sharp procedure: The 5′ termini of 15S β‐globin mRNA and mature 10S β‐globin mRNA have identical map coordinates. Nucl. Acids Res. 7:1175‐1193.
   Zinn, K., DiMaio, D., and Maniatis, T. 1983. Identification of two distinct regulatory regions adjacent to the human β‐interferon gene. Cell 34:865‐879.
Key References
   Chamberlin and Ryan 1982. See above.
  Summarizes properties of bacteriophage RNA polymerases and provides references to the original literature.
   Melton et al., 1984. See above.
  Describes the construction of vectors carrying promoters for SP6 RNA polymerase, use of the polymerase for preparing RNA probes and large quantities of biologically active RNA, and biochemical properties of the transcription reaction.
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