Chapter 18 - Quantification of Specific mRNAs by Nuclease Protection

Abstract

The various permutations of polymerase chain reaction (PCR) offer the most compelling quantitative expression profile of a sample. A variety of nucleases and approaches have been used to quantify and characterize specific transcripts in greater detail than can be derived from Northern analysis in a non-PCR format. The basic methodology of nuclease protection accommodates denatured double-stranded DNA probes as well as RNA probes. These techniques include primer extension, the nuclear runoff assay, the ribonuclease protection assay and the S1 nuclease protection assay. In the S1 nuclease assay for the quantification of specific RNA species, purified RNA is hybridized in solution with a labeled probe sequence to form thermodynamically stable hybrid molecules. Any 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 protected RNAs are then deduced by autoradiography, performed directly from the gel. Whereas in case of 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 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 key advantage that these techniques offer is that they do not require a reverse transcriptase step. Further, because there is no filter-based immobilization on nucleic acid molecules (as in Northern blots), sensitivity is enhanced.