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‚Äźhybridized with a different probe.

     
 
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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
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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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 ( 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 5.7.1   MaterialsSelection of Cloning Vectors Incorporating Promoters for Bacteriophage RNA Polymerases   Properties of Materials Used for Immobilization of Nucleic Acids b   Properties of Materials Used for Immobilization of Nucleic Acids

    Vector Size (bp) Markers a Promoters
    pBluescript 2950 amp, lacZ T3, T7
    pGEM series 2746‐3223 amp, lacZ SP6, T7
    pGEMEX‐1 4200 amp SP6, T3, T7
    pSELECT‐1 3422 tet, lacZ SP6, T7
    pSP18, 19, 64, 65 2999‐3010 amp SP6
    pSP70, 71, 72, 73 2417‐2464 amp SP6, T7
    pSPORT1 4109 amp, lacZ SP6, T7
    pT3/T7 series 2700, 2950 amp, lacZ T3, T7
    pWE15 8800 amp, neo T3, T7
    pWE16 8800 amp, dhfr T3, T7
    Nitrocellulose Supported nitrocellulose Uncharged nylon Positively charged nylon Activated papers
    Application ssDNA, RNA, protein ssDNA, RNA, protein ssDNA, dsDNA, DNA, protein ssDNA, dsDNA, RNA, protein ssDNA, RNA
    Binding capacity (µg nucleic acid/cm2) 80‐100 80‐100 400‐600 400‐600 2‐40
    Tensile strength Poor Good Good Good Good
    Mode of nucleic acid attachment c Noncovalent Noncovalent Covalent Covalent Covalent
    Lower size limit for efficient nucleic acid retention 500 nt 500 nt 50 nt or bp 50 nt or bp 5 nt
    Suitability for reprobing Poor (fragile) Poor (loss of signal) Good Good Good
    Commercial examples Schleicher & 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

     aAbbreviations: amp, ampicillin resistance; dhfr, dihydrofolate reductase; lacZ, β‐galactosidase α‐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 5.7.2   MaterialsSelection of Cloning Vectors Incorporating Promoters for Bacteriophage RNA Polymerases   Properties of Materials Used for Immobilization of Nucleic Acids b   Properties of Materials Used for Immobilization of Nucleic Acids

    Vector Size (bp) Markers a Promoters
    pBluescript 2950 amp, lacZ T3, T7
    pGEM series 2746‐3223 amp, lacZ SP6, T7
    pGEMEX‐1 4200 amp SP6, T3, T7
    pSELECT‐1 3422 tet, lacZ SP6, T7
    pSP18, 19, 64, 65 2999‐3010 amp SP6
    pSP70, 71, 72, 73 2417‐2464 amp SP6, T7
    pSPORT1 4109 amp, lacZ SP6, T7
    pT3/T7 series 2700, 2950 amp, lacZ T3, T7
    pWE15 8800 amp, neo T3, T7
    pWE16 8800 amp, dhfr T3, T7
    Nitrocellulose Supported nitrocellulose Uncharged nylon Positively charged nylon Activated papers
    Application ssDNA, RNA, protein ssDNA, RNA, protein ssDNA, dsDNA, DNA, protein ssDNA, dsDNA, RNA, protein ssDNA, RNA
    Binding capacity (µg nucleic acid/cm2) 80‐100 80‐100 400‐600 400‐600 2‐40
    Tensile strength Poor Good Good Good Good
    Mode of nucleic acid attachment c Noncovalent Noncovalent Covalent Covalent Covalent
    Lower size limit for efficient nucleic acid retention 500 nt 500 nt 50 nt or bp 50 nt or bp 5 nt
    Suitability for reprobing Poor (fragile) Poor (loss of signal) Good Good Good
    Commercial examples Schleicher & 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

     bThis table is based on Brown ( ), with permission from BIOS Scientific Publishers Ltd.
     cAfter 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 and appendix 1A in this manual), and autoradiography (CPMB APPENDIX 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

  • 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

  • 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 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 (see CPMB APPENDIX 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

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.
   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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