Analysis of RNA by Northern Blot Hybridization

Terry Brown1, Karol Mackey2

1 University of Manchester Institute of Science and Technology, Manchester, 2 Molecular Research, Cincinnati, Ohio
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3K
DOI:  10.1002/0471142905.hga03ks30
Online Posting Date:  November, 2001
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Abstract

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Because they are single‐stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. The describes blotting and hybridization of RNA fractionated in an agarose‐formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot‐blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a .

     
 
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Table of Contents

  • Basic Protocol 1: Northern Hybridization of RNA Fractionated by Agarose‐Formaldehyde Gel Electrophoresis
  • Support Protocol 1: Removal of Probes From Northern Blots
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Northern Hybridization of RNA Fractionated by Agarose‐Formaldehyde Gel Electrophoresis

  Materials
  • recipe10× and 1× MOPS running buffer (see recipe for 10× buffer)
  • 12.3 M (37%) formaldehyde, pH >4.0
  • RNA sample: total cellular RNA (unit 10.4) or poly(A)+ RNA (e.g., CPMB UNIT )
  • Formamide
  • recipeFormaldehyde loading buffer (see recipe)
  • 0.5 M ammonium acetate and 0.5 µg/ml ethidium bromide in 0.5 M ammonium acetate or recipe10 mM sodium phosphate (pH 7.0; see recipe)/1.1 M formaldehyde with and without 10 µg/ml acridine orange
  • 0.05 M NaOH/1.5 M NaCl (optional)
  • 0.5 M Tris⋅Cl (pH 7.4; appendix 2D)/1.5 M NaCl (optional)
  • 20×, 2×, and 6× SSC ( appendix 2D)
  • 0.03% (w/v) methylene blue in 0.3 M sodium acetate, pH 5.2 (optional)
  • DNA suitable for use as probe or for in vitro transcription to make RNA probe
  • recipeFormamide prehybridization/hybridization solution (see recipe)
  • 2× SSC/0.1% (w/v) SDS
  • 0.2× SSC/0.1% (w/v) SDS, room temperature and 42°C
  • 0.1× SSC/0.1% (w/v) SDS, 68°C
  • 55°, 60°, and 100°C water baths
  • Oblong sponge slightly larger than the gel being blotted
  • RNase‐free glass dishes ( appendix 2A)
  • Whatman 3MM filter paper sheets
  • UV‐transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)
  • Nitrocellulose or nylon membrane
  • Glass plate of appropriate size
  • Vacuum oven
  • UV transilluminator, calibrated (e.g.,CPMB UNIT )
  • Hybridization oven (e.g., Hybridiser HB‐1, Techne) and tubes
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), radiolabeling of DNA by nick translation or random oligonucleotide priming ( appendix 3E), RNA labeling by in vitro synthesis (unit 7.2), measuring specific activity of labeled nucleic acids and separating unincorporated nucleotides from labeled nucleic acids ( appendix 3.)
NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in appendix 2A.

Support Protocol 1: Removal of Probes From Northern Blots

  Materials
  • Northern hybridization membrane containing probe (see protocol 1)
  • recipeStripping solution (see recipe)
  • Hybridization bags
  • 65°, 80°, or 100°C (boiling) water bath
  • UV‐transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)
CAUTION: If hybridization probes include a radioactive label, dispose of stripping solutions as radioactive waste. Observe appropriate caution when working with the toxic compound formamide.
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Figures

Videos

Literature Cited

Literature Cited
   Alwine, J.C., Kemp, D.J., and Stark, G.R. 1977. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl‐paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. U.S.A. 74:5350‐5354.
   Bailey, J.M. and Davidson, N. 1976. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis Anal. Biochem. 70:75‐85.
   Bodkin, D.K. and Knudson, D.L. 1985. Assessment of sequence relatedness of double‐stranded RNA genes by RNA‐RNA blot hybridization. J. Virol. Methods 10:45‐52.
   Casey, J. and Davidson, N. 1977. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucl. Acids Res. 4:1539‐1552.
   Chomczynski, P. 1992. One‐hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201:134‐139.
   Herrin, D.L. and Schmidt, G.W. 1988. Rapid, reversible staining of Northern blots prior to hybridization BioTechniques. 6:196‐200.
   Lehrach, H., Diamond, D., Wozney, J.M., and Boedtker, H. 1977. RNA molecular weight determinations by gel electrophoresis under denaturing conditions: A critical reexamination. Biochemistry 16:4743‐4751.
   Peferoen, M., Huybrechts, R., and De Loof, A. 1982. Vacuum‐blotting: A new simple and efficient transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to nitrocellulose. FEBS Lett. 145:369‐372.
   Smith, M.R., Devine, C.S., Cohn, S.M., and Lieberman, M.W. 1984. Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose. Anal. Biochem. 137:120‐124.
   Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis J. Mol. Biol. 98:503‐517.
   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.
   Wilkinson, M. 2000. Purification of RNA. In Essential Molecular Biology: A Practical Approach, Vol. 1 (T.A. Brown, ed.) pp. 69‐88. Oxford University Press, Oxford.
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