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. 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.