Ribonuclease Protection Analysis of Gene Expression in Xenopus

When characterizing the developmental expression of a novel gene, or when examining the response of a known gene to experimental manipulations, it is important to be able to assay mRNA transcript levels accurately. Although a number of techniques for transcript analysis are available, one of the most useful and widespread is the ribonuclease (RNase) protection assay (for example, see Fig. 1 ). The major advantages of RNase protection analysis are good sensitivity, excellent specificity, and the linear response to differing transcript levels. Perhaps the major disadvantage of RNase protection is the need to prepare special RNA probes and the fact that the RNA samples used for RNase protection analysis are destroyed and therefore cannot be reused. In addition to expression analysis, RNase protection can also be applied to a number of additional experimental goals, including mapping of transcription start sites, mapping of intron/exon boundaries, analysis of alternative splicing, and determination of the rate of degradation of nucleic acids introduced into the embryo.