Analysis of RNA by Northern and Slot‐Blot Hybridization

Terry Brown1, Karol Mackey2

1 University of Manchester Institute of Science and Technology, Manchester, United Kingdom, 2 Molecular Research, Cincinnati, Ohio
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.17
DOI:  10.1002/0471142301.ns0517s15
Online Posting Date:  August, 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), while unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Included in this unit are detailed procedures for RNA denaturation, blotting and hybridization. Also described is a method for stripped hybridization probes from blots so the blots can be re-hybridized with a different probe.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: 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: Removal of Probes from Northern Blots
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: 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 (appendix 1I) or poly(A)+ RNA (appendix 1I)
  • Formamide
  • Formaldehyde loading buffer (see recipe)
  • 0.5 M ammonium acetate and 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
  • 0.24- to 9.5-kb RNA ladder (Life Technologies) (optional)
  • 0.05 M NaOH/1.5 M NaCl (optional)
  • 0.5 M Tris×Cl (pH 7.4; appendix 2A)/1.5 M NaCl (optional)
  • 20×, 2×, and 6× SSC (appendix 2A)
  • 0.03% (w/v) methylene blue in 0.3 M sodium acetate, pH 5.2 (optional)
  • DNA suitable for use as probe (unit 5.1) or for in vitro transcription to make RNA probe (Table 5.17.1)
     
    Table 5.17.1 Selection of Cloning Vectors Incorporating Promoters for Bacteriophage RNA Polymerases
    VectorSize (bp)MarkersaPromoters
    pBluescript2950amp, lacZ¢T3, T7
    pGEM series2746-3223amp, lacZ¢SP6, T7
    pGEMEX-14200ampSP6, T3, T7
    pSELECT-13422tet, lacZ¢SP6, T7
    pSP18, 19, 64, 652999-3010ampSP6
    pSP70, 71, 72, 732417-2464ampSP6, T7
    pSPORT14109amp, lacZ¢SP6, T7
    pT3/T7 series2700, 2950amp, lacZ¢T3, T7
    pWE158800amp, neoT3, T7
    pWE168800amp, dhfrT3, T7
     a Abbreviations: amp, ampicillin resistance; dhfr, dihydrofolate reductase; lacZ, -galactosidase -peptide; neo, neomycin phosphotransferase (kanamycin resistance); tet, tetracycline resistance.
  • Formamide 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 (baked for 4 hr at 300°C)
  • Whatman 3MM filter paper sheets
  • UV-transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)
  • Nitrocellulose or nylon membrane (see Table 5.17.2 for list of suppliers)
     
    Table 5.17.2 Properties of Materials Used for Immobilization of Nucleic Acids a
    NitrocelluloseSupported nitrocelluloseUncharged nylonPositively charged nylonActivated papers
    ApplicationssDNA, RNA, proteinssDNA, RNA, proteinssDNA, dsDNA, DNA, proteinssDNA, dsDNA, RNA, proteinssDNA, RNA
    Binding capacity (µg nucleic acid/cm2)80-10080-100400-600400-6002-40
    Tensile strengthPoorGoodGoodGoodGood
    Mode of nucleic acid attachmentbNoncovalentNoncovalentCovalentCovalentCovalent
    Lower size limit for efficient nucleic acid retention500 nt500 nt50 nt or bp50 nt or bp5 nt
    Suitability for reprobingPoor (fragile)Poor (loss of signal)GoodGoodGood
    Commercial examplesSchleicher & Schuell BA83, BA85;

    Amersham Hybond-C    		Schleicher & Schuell Optibond;

    Amersham Hybond-C extra    		Amersham Hybond-N;

    Stratagene Duralon-UV;

    Du Pont NEN GeneScreen    		Schleicher & Schuell Nytran;

    Amersham Hybond-N+;

    Bio-Rad ZetaProbe;

    PALL Biodyne B;

    Du Pont NEN GeneScreen Plus    		Schleicher & Schuell APT papers
     a This table is based on Brown (1991), with permission from BIOS Scientific Publishers Ltd.
     b After suitbale immobilization procedure (see text).
  • Glass plate slightly larger than final membrane size
  • Vacuum oven
  • UV transilluminator, calibrated
  • Hybridization oven (e.g., Hybridiser HB-1, Techne) and tubes
  • Additional reagents and equipment for agarose gel electrophoresis (appendix 1N), radiolabeling of DNA by nick translation or random oligonucleotide priming (unit 4.2), RNA labeling by in vitro synthesis (unit 4.3), measuring specific activity of labeled nucleic acids and separating unincorporated nucleotides from labeled nucleic acids (cpmb unit 3.4 and appendix 1A in this manual), and autoradiography (cpmb appendix 3A and appendix 1A in this manual)

NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in appendix 2A; 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 (also see Basic Protocol)
  • 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 2A)
  • 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 appendix 2A 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 (also see Basic Protocol)
  • 0.1 M NaOH
  • 10× SSC (appendix 2A)
  • 20× SSC, 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 appendix 2A; see unit introduction for further instructions and precautions regarding establishment of an RNase-free environment.

Support Protocol: Removal of Probes from Northern Blots

 Materials
  • Northern hybridization membrane containing probe (see Basic Protocol, Alternate Protocol 1, or Alternate Protocol 2)
  • 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 (see cpmb appendix 3A and appendix 1A in this manual)

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

  •  FigureFigure 5.17.1 Rat liver RNA (5 µg) was electrophoresed on a formaldehyde 1% agarose gel containing ethidium bromide (left), transferred to a hybridization membrane and stained with methylene blue stain (Molecular Research Center; Herrin and Schmidt, 1988, right). Shown are 28S (4.7 kb) and 18S (1.9 kb) ribosomal RNAs, as well as 4S to 5S (0.10 to 0.15 kb) RNA containing mix of tRNA and 5S ribosomal RNA.
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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.
    Brown, T.A. (ed.) 1991. Molecular Biology Labfax. BIOS Scientific Publishers, Oxford.
    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. 2000. Purification of RNA. In Essential Molecular Biology:A Practical Approach 2nd edition., Vol. 1 (T.A. Brown, ed.) pp. 69-88. Oxford University Press, Oxford.
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