Analysis of RNA by Northern and Slot‐Blot Hybridization

Terry Brown1

1 University of Manchester Institute of Science and Technology, Manchester, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.12
DOI:  10.1002/0471142735.im1012s07
Online Posting Date:  May, 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. The in this unit describes blotting and hybridization of RNA fractionated in an agarose/formaldehyde gel. This is arguably the quickest and most reliable method for northern analysis of specific sequences in RNA extracted from eukaryotic cells. An alternate protocol gives details of the glyoxal/DMSO method for denaturing gel electrophoresis, which may provide better resolution of some RNA molecules. A second describes slotā€blot hybridization of RNA samples, a rapid method for assessing the relative abundance of an RNA species in extracts from different tissues.

     
 
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Table of Contents

  • Basic Protocol 1: Northern Hybridization of RNA Fractionated by Agarose/Formaldehyde Gel Electrophoresis
  • Alternate Protocol 1: Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment
  • Alternate Protocol 2: Northern Hybridization of Unfractionated RNA Immobilized by Slot Blotting
  • 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
  • Diethylpyrocarbonate (DEPC)
  • recipe10× and 1× MOPS running buffer
  • 12.3 M (37% v/v) formaldehyde, pH >4.0
  • RNA sample: total cellular RNA or poly(A)+ RNA (unit 10.11)
  • Formamide
  • recipeFormaldehyde loading buffer
  • 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; reagents and solutions)/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)/ 1.5 M NaCl (optional)
  • recipe20×, 2×, and 6× SSC (unit 10.6)
  • 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 (Table 97.80.4711)
  • Formamide prehybridization/hybridization solution (unit 10.6)
  • recipe2× SSC/0.1% (w/v) SDS
  • recipe0.2× SSC/0.1% (w/v) SDS, room temperature and 42°C
  • 0.1× SSC/0.1% (w/v) SDS, 68°C
  • 60°C water bath
  • Oblong sponge slightly larger than the gel being blotted
  • Whatman 3MM filter paper sheets
  • Nitrocellulose or nylon membrane (see Table 97.80.4711 for list of suppliers)
  • UV transilluminator, calibrated (unit 10.6)
  • UV‐transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)
  • Hybridization oven (e.g., Hybridiser HB‐1, Techne)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.6), radiolabeling of DNA by nick translation or random oligonucleotide priming and RNA labeling by in vitro synthesis (unit 10.10), measuring specific activity of labeled nucleic acids and separating unincorporated nucleotides from labeled nucleic acids (unit 10.9), and autoradiography ( appendix 3A)
NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in unit 10.11; see unit introduction for further instructions and precautions regarding establishment of an RNase‐free environment.

Alternate Protocol 1: Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment

  Additional Materials
  • recipe10 mM and 100 mM sodium phosphate, pH 7.0
  • Dimethyl sulfoxide (DMSO)
  • recipe6 M (40% v/v) glyoxal, deionized immediately before use ( reagents and solutions)
  • recipeGlyoxal loading buffer
  • 20 mM Tris⋅Cl, pH 8.0
  • Apparatus for recirculating running buffer during electrophoresis
NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in unit 10.11; see unit introduction for further instructions and precautions regarding establishment of an RNase‐free environment.

Alternate Protocol 2: Northern Hybridization of Unfractionated RNA Immobilized by Slot Blotting

  Additional Materials
  • 0.1 M NaOH
  • recipe10× SSC (unit 10.6)
  • recipe20× SSC, room temperature and ice cold
  • recipeDenaturing solution
  • recipe100 mM sodium phosphate, pH 7.0
  • Dimethyl sulfoxide (DMSO)
  • recipe6 M (40% v/v) glyoxal, deionized immediately before use ( reagents and solutions)
  • Manifold apparatus with a filtration template for slot blots (e.g., Bio‐Rad Bio‐Dot SF, Schleicher and Schuell Minifold II)
NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in unit 10.11; see unit introduction for further instructions and precautions regarding establishment of an RNase‐free environment.
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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.
   Kafatos, F.C., Jones, C.W., and Efstratiadis, A. 1979. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure Nucl. Acids Res. 7:1541‐1552.
   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 (Fed. Eur. Biochem. Soc.) 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. 1991. Purification of RNA. In Essential Molecular Biology: A Practical Approach, Vol. 1 (T.A. Brown, ed.) pp.69‐87. IRL Press at Oxford University Press, Oxford.
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