Solution hybridization-nuclease protection assays for sensitive detection of differentially spliced substance P- and...

In this chapter we discussed methods that can be used for the sensitive detection and quantitation of differentially or alternatively spliced mRNAs as well as mRNAs of low abundance. Although mechanisms responsible for splicing (and differential splicing in particular) have not been fully determined, many RNAs derived from a variety of genes have been observed to undergo the process. The impact of splicing with regard to the expanded potential of gene expression emphasizes the usefulness of the solution hybridization-nuclease digestion technique described here, compared to Northern blot analysis. The use of radiolabeled cRNA(s) provides for an assay of both high specificity and high sensitivity. While end-labeled cDNA probes can be used, they do not have the sensitivity inherent in the assay performed with uniformly radiolabeled cRNAs. If multiple mRNAs are derived from a single gene as a result of differential or alternative precursor RNA splicing, however, the results with a cRNA probe may initially appear to be quite complicated, and end-labeled cDNAs may yield more easily interpretable results. Nonetheless, both types of probes are useful in the context of gene expression analysis, and it is clear that for routine purposes of quantitation cRNA probes in solution hybridization-nuclease protection assays are clearly more desirable than RNA blot analyses due to their truly quantitative nature as well as ease of assay.