Stripping hybridization probes from blots can be done under three different sets of conditions these methods are outlined in a Support Protocol. Several methods have been described for measuring the level of expression of a gene in a tissue.
#NORTHERN BLOT ANALYSIS OF RNA PDF#
Alternate Protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Analysis of RNA by Northern Blotting Using Riboprobes Authors: Rai Ajit Srivastava 1 Show more details PDF Full Text Abstract In studying the expression of a gene in mammalian tissues, it is important to determine the levels of the corresponding mRNA. The Basic Protocol describes blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. 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. 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. Northern blotting differs from Southern blotting largely in the initial gel fractionation step. In the present immuno-northern blot analysis (described in detail in Materials and Methods), RNAs are detected by antibodies against the modified nucleosides instead of by the radio-labelled DNA probes used for a conventional northern blot protocol. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Immuno-northern blotting using antibodies against modified nucleosides. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting) unfractionated RNA is immobilized by slot or dot blotting. Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA.
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