RNA pp 87-105 | Cite as

Northern Blotting Analysis

Part of the Methods in Molecular Biology book series (MIMB, volume 703)


Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membrane is analysed by hybridization to one or more specific probes that are labelled for subsequent detection. Northern blotting is relatively simple to perform, inexpensive, and not plagued by artefacts. Recent developments of hybridization membranes and buffers have resulted in increased sensitivity closing the gap to the more laborious nuclease protection experiments.

Key words

Gel electrophoresis northern blotting probe hybridization analysis mRNA 


  1. 1.
    Alwine, J. C., Kemp, D. J., 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 USA 74, 5350–5354.CrossRefPubMedGoogle Scholar
  2. 2.
    Alwine, J. C., Kemp, D. J., Parker, B. A., Reiser, J., Renart, J., Stark, G. R., Wahl, G. M. (1979) Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Methods Enzymol 68, 220–242.CrossRefPubMedGoogle Scholar
  3. 3.
    Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503–517.CrossRefPubMedGoogle Scholar
  4. 4.
    Lamond, A. I., Sproat, B. S. (1994) Isolation and characterization of Ribonucleoprotein complexes, in (Higgins, S. J. and Hames, B. D., eds.) RNA Processing. A Practical Approach. IRL Press, Oxford, Vol. 1, pp. 103–140.Google Scholar
  5. 5.
    Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  6. 6.
    Darling, D. C., Brickell, P. M. (1994) Nucleic Acid Blotting. The Basics.. IRL Press, Oxford.Google Scholar
  7. 7.
    Farrell, R. E.,Jr. (1993) RNA Methodologies. A Laboratory Guide for Isolation and Characterization. Academic, San Diego, CA.Google Scholar
  8. 8.
    Chomczynski, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156–159.CrossRefPubMedGoogle Scholar
  9. 9.
    Aviv, H., Leder, P. (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci USA 69, 1408–1412.CrossRefPubMedGoogle Scholar
  10. 10.
    Lehrach, H., Diamond, D., Wozney, J. M., Boedtker, H. (1977) RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16, 4743–4751.CrossRefPubMedGoogle Scholar
  11. 11.
    McMaster, G. K., Carmichael, G. G. (1977) Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74, 4835–4838.CrossRefPubMedGoogle Scholar
  12. 12.
    Reijnders, L., Sloof, P., Sival, J., Borst, P. (1973) Gel electrophoresis of RNA under denaturing conditions. Biochim Biophys Acta 324, 320–333.PubMedGoogle Scholar
  13. 13.
    Chomczynski, P., Mackey, K. (1994) One-hour downward capillary blotting of RNA at neutral pH. Anal Biochem 221, 303–305.CrossRefPubMedGoogle Scholar
  14. 14.
    Bittner, M., Kupferer, P., Morris, C. F. (1980) Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Anal Biochem 102, 459–471.CrossRefPubMedGoogle Scholar
  15. 15.
    Herrin, D. L., Schmidt, G. W. (1988) Rapid, reversible staining of northern blots prior to hybridization. Biotechniques 6, 196–200.PubMedGoogle Scholar
  16. 16.
    Church, G. M., Gilbert, W. (1984) Genomic sequencing. Proc Natl Acad Sci USA 81, 1991–1995.CrossRefPubMedGoogle Scholar
  17. 17.
    Saito, I., Sugiyama, H., Furukawa, N., Matsuura, T.. (1981) Photoreaction of thymidine with primary amines. Application to specific modification of DNA. Nucleic Acids Symp Ser 10, 61–64.PubMedGoogle Scholar
  18. 18.
    Feinberg, A. P., Vogelstein, B.. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6–13.CrossRefPubMedGoogle Scholar
  19. 19.
    Casey, J., Davidson, N. (1977) Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res 4, 1539–1552.CrossRefPubMedGoogle Scholar
  20. 20.
    Bonner, J., Kung, G., Bekhor, I. (1967) A method for the hybridization of nucleic acid molecules at low temperature. Biochemistry 6, 3650–3653.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.The Bartholin Institute, Copenhagen University HospitalCopenhagenDenmark
  2. 2.Department of Cellular and Molecular MedicineThe Panum Institute, University of CopenhagenCopenhagenDenmark

Personalised recommendations