Northern blotting.

Abstract

Northern blotting is the technique whereby RNA molecules are denatured, fractionated through agarose on the basis of size, and then transferred to a solid matrix for subsequent hybridization to a specific labeled probe. The technique can be used to analyze the pattern of transgene expression in an organism, and changes in that pattern following physiological and developmental transitions. It is important to note that the technique depends on being able to distinguish the transgene RNA from the RNAs produced by the homologous endogenous gene. Thus, the transgene must be modified such that, although both transgene and endogenous RNAs are detected with the same probe, the former RNAs have a different mobility to the latter. Alternatively, the transgene RNAs should contain hybridizable segments with little or no homology to host RNAs expressed in the tissues being examined-for example, RNAs derived from a hybrid gene with a viral or prokaryotic reporter element or a gene from another species with sufficient sequence divergence to allow the transgene RNAs to be distinguished from the host RNAs using specific probes.