Hybridization and Detection of Digoxigenin Probes on RNA Blots

  • Elizabeth Davies
  • Rachel Hodge
  • Peter G. Isaac
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 28)

Abstract

The probing of RNA gel blots (also called Northern blots) with labeled nucleic acids provides data on the relative levels of steady state gene expression, and on RNA processing. The principle of the technique was first described by Alwine et al. (1). The protocol described in this chapter demonstrates the use of nonradioactive digoxigenin-labeled probes ( chapter 10 and  chapter 11) on RNA blots ( chapter 7 and  chapter 8). The detection system employs an antidigoxigenin antibody/alkaline phosphatase conjugate, and the chemiluminescent substrate 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) (2,3). The procedure used is similar to that described for probing Southern blots and detection by chemiluminescence in  chapter 17. However, this procedure uses a different membrane for support of the immobilized nucleic acids. This, and the fact that RNA is being probed, require modifications to the hybridization buffer and washing procedure.

Keywords

Citrate MgCl2 Dodecyl Formamide Lauryl 

References

  1. 1.
    Alwine, J. C., Kemp, D. J., and Stark, G. R. (1977) Method for detection of specific RNA in agarose gels by transfer to diazo benzyloxymethyl paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350–5354.PubMedCrossRefGoogle Scholar
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    Voyta, J. C., Edwards, B., and Bronstein, I. (1988) Ultrasensitive chemiluminescent detection of alkaline phosphatase activity. Clin. Chem. 34, 1157.Google Scholar
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    Bronstein, I., Voyta, J. C., and Edwards, B. (1989) A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay. Analyt. Biochem. 180, 95–98.PubMedCrossRefGoogle Scholar
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    Paul, W., Hodge, R., Smartt, S., Draper, J., and Scott, R. (1992) The isolation and characterisation of the tapetum-specific Arabidopsis A9 gene. Plant Mol. Biol. 19, 611–622.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1994

Authors and Affiliations

  • Elizabeth Davies
    • 1
  • Rachel Hodge
    • 2
  • Peter G. Isaac
    • 1
  1. 1.Nickerson BIOCEM Ltd.CambridgeUK
  2. 2.Department of BotanyUK

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