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Northern Analysis: The Basics


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Steps > RNA Isolation, Probe Generation > Electrophoresis, Transfer > Hybridization, Detection > Stripping & Reprobing


Northern analysis remains a standard method for detection and quantitation of mRNA levels despite the advent of powerful techniques, such as RT-PCR, gene array analysis and nuclease protection assays. Northern analysis provides a direct relative comparison of message abundance between samples on a single membrane. It is the preferred method for determining transcript size and for detecting alternatively spliced transcripts.

The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., integrity of the RNA sample and how efficiently it has transferred to the membrane). RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. Northern hybridization is exceptionally versatile in that radiolabeled or nonisotopically labeled DNA, in vitro transcribed RNA and oligonucleotides can all be used as hybridization probes. Additionally, sequences with only partial homology (e
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