Northern hybridization: rapid and simple electrophoretic conditions.

Current methods for Northern blot analysis to determine mRNA abundance in tissues are tedious and utilize toxic chemicals. The requirement for chemicals of high purity, free of ribonucleases, and the lability of RNA, in comparison to DNA, are additional factors preventing wider use of the technique. For Northern blot analysis, RNA is generally denatured with glyoxal (1) or formaldehyde (2) prior to electrophoresis in agarose gels. The gels require extensive washing for ethidium bromide or acridine orange staining to localize major RNA species or fragments used as size markers. These manipulations are time-consuming and contain several steps where ribonucleases can be introduced, which can result in failure of the experiment. In addition, these techniques do not lend themselves to easy visualization of the degree of RNA migration during electrophoresis or allow the experimenter to determine the efficiency of transfer of the RNA following blotting. This report outlines a rapid and simple method which requires just a 5 minute denaturation of RNA samples in loading buffer and allows the experimenter to monitor the integrity and migration of major RNA species and size markers during electrophoresis (Figure 1A). In addition, the stained RNA is easily detected following blotting onto a nylon or nitrocellulose membrane, thus allowing the experimenter to determine the efficiency of transfer of the RNA from the gel (Figure IB). The technique is summarized in the following three easy steps.