UNIT 10.12 Analysis of RNA by Northern and Slot-Blot Hybridization

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. The Basic Protocol in this unit describes blotting and hybridization of RNA fractionated in an agarose/formaldehyde gel. This is arguably the quickest and most reliable method for northern analysis of specific sequences in RNA extracted from eukaryotic cells. An alternate protocol gives details of the glyoxal/DMSO method for denaturing gel electrophoresis, which may provide better resolution of some RNA molecules. A second Alternate Protocol describes slot-blot hybridization of RNA samples, a rapid method for assessing the relative abundance of an RNA species in extracts from different tissues.