Chapter 18 - Quantification of specific mRNAs by nuclease protection

Abstract

A variety of non-PCR assays are used to measure the abundance of specific transcripts in greater detail than is possible with Northern analysis. Among these techniques are the ribonuclease protection assay and the S1 nuclease protection assay, generically known as a nuclease protection assay. In the S1 nuclease assay for gene expression analysis, purified RNA is hybridized in solution with a labeled probe sequence to form thermodynamically stable hybrid molecules, also known as protected fragments. Any sample RNA or probe molecules that do not participate in the formation of hybrid molecules are digested away by the single-strand-specific nuclease S1, followed by electrophoresis of the intact hybrid molecules. The size and abundance of the protected RNAs are then deduced by autoradiography, performed directly from the gel. In the case of the RNase protection assay for the quantification of specific RNA species, purified RNA is hybridized in solution with a labeled antisense probe sequence to form thermodynamically stable double-stranded RNA molecules. Any sample RNA or probe molecules that remain single-stranded are digested by an RNase cocktail. Following electrophoresis, the size and abundance of protected RNAs are then deduced by autoradiography, performed directly from the gel. The elimination of the blotting step and sample immobilization on a membrane (as in performed in Northern analysis) greatly improves the sensitivity of these assays.