Analysis of RNA by Northern and Slot Blot Hybridization

Terry Brown1, Karol Mackey2, Tingting Du3

1 University of Manchester Institute of Science and Technology, Manchester, 2 Molecular Research, Cincinnati, Ohio, 3 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.9
DOI:  10.1002/0471142727.mb0409s67
Online Posting Date:  September, 2004
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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. Northern blotting differs from Southern blotting largely in the initial gel fractionation step. 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. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol 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 Support Protocol.

     
 
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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
  • Support Protocol 1: Removal of Probes from Northern Blots
  • Basic Protocol 2: Northern Hybridization of Small RNA Fractionated by Polyacrylamide Gel Electrophoresis
  • Alternate Protocol 3: Northern Hybridization of RNA Using Church's Hybridization Buffer
  • 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
  • 10× and 1× MOPS running buffer (see recipe for 10× buffer)
  • 12.3 M (37%) formaldehyde, pH >4.0
  • RNA sample: total cellular RNA (units 4.1 4.4) or poly(A)+ RNA (unit 4.5)
  • Formamide
  • Formaldehyde loading buffer (see recipe)
  • 0.5 µg/ml ethidium bromide in 0.5 M ammonium acetate or 10 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 22)/1.5 M NaCl (optional)
  • 20×, 2×, and 6× SSC ( appendix 22)
  • 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 2.10)
  • 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 (unit 4.1)
  • Whatman 3MM filter paper sheets
  • UV‐transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)
  • Nitrocellulose or nylon membrane (see Table 97.80.4711 for list of suppliers)
  • Glass plate of appropriate size (Fig. )
  • Vacuum oven
  • UV transilluminator, calibrated (unit 2.9)
  • Hybridization oven (e.g., Hybridiser HB‐1, Techne) and tubes
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5), radiolabeling of DNA by nick translation or random oligonucleotide priming (unit 3.5), RNA labeling by in vitro synthesis (unit 2.10), measuring specific activity of labeled nucleic acids and separating unincorporated nucleotides from labeled nucleic acids (unit 3.4), and autoradiography ( appendix 3A)
NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in unit 4.1; 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

  • 10 mM and 100 mM sodium phosphate, pH 7.0 (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • 6 M (40%) glyoxal, deionized immediately before use (see recipe)
  • Glyoxal loading buffer (see recipe)
  • 20 mM Tris·Cl, pH 8.0 ( appendix 22)
  • Apparatus for recirculating running buffer during electrophoresis
  • 50° and 65°C water baths
NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in unit 4.1; 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

  • 0.1 M NaOH
  • 10× SSC ( appendix 22)
  • 20× SSC ( appendix 22), room temperature and ice‐cold
  • Denaturing solution (see recipe)
  • 100 mM sodium phosphate, pH 7.0 (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • 6 M (40%) glyoxal, deionized immediately before use (see recipe)
  • Manifold apparatus with a filtration template for slot blots (e.g., Bio‐Rad Bio‐Dot SF, Schleicher and Schuell Minifold II)
  • 50° and 60°C water baths
NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in unit 4.1; see unit introduction for further instructions and precautions regarding establishment of an RNase‐free environment.

Support Protocol 1: Removal of Probes from Northern Blots

  Materials
  • Northern hybridization membrane containing probe (see protocol 1, protocol 2, or protocol 3)
  • Stripping solution (see recipe)
  • Hybridization bags
  • 65°, 80°, or 100° (boiling) water bath
  • UV‐transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)
  • Additional reagents and equipment for autoradiography ( appendix 3A)
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.

Basic Protocol 2: Northern Hybridization of Small RNA Fractionated by Polyacrylamide Gel Electrophoresis

  • Church's hybridization buffer without BSA (see recipe)
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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 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, Oxford.
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