The RNase Protection Assay

The RNase protection assay is based on the resistance of RNA:RNA hybrids to single-strand specific RNases, after annealing to a complementary ³² P-labeled probe in solution. It can be used to map the ends of RNA molecules or exon-intron boundaries. It also provides an attractive and highly sensitive alternative to Northern blot hybridization for the quantitative determination of mRNA abundance. Hybridization is carried out with an excess concentration of probe so that all complementary sequences are driven into the labeled hybrid. Unhybridized probe or any single-stranded regions of the hybridized probe are then removed by RNase digestion. The “protected” probe is detected and quantitated on a denaturing polyacrylamide gel. Originally, single-stranded DNA probes were used for the assay (1 ). However, these have the disadvantage of being lengthy to prepare. The ease with which labeled RNA molelcules (riboprobes; see Chapter 12 ) can now be made makes an assay based on RNA RNA hybridization much more favorable (2 ,3 ).