Analysis of RNA by Northern 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. 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. 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.