Unit

UNIT 4.9 Analysis of RNA by Northern and Slot Blot Hybridization

  1. Terry Brown1,
  2. Karol Mackey (probe removal)2

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb0409s37

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Brown, T. and Mackey, K. 2001. Analysis of RNA by Northern and Slot Blot Hybridization. Current Protocols in Molecular Biology. 4:II:4.9.

Author Information

  1. 1

    University of Manchester Institute of Science and Technology, Manchester, United Kingdom

  2. 2

    Molecular Research, Cincinnati, Ohio

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JAN 1997

This is not the most recent version of the article. View current version (1 SEP 2004)

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.