Quantitative Measurement of mRNA Using the RNase Protection Assay

RNase protection provides a sensitive method for detecting and quantitating specific RNAs. The method relies on the ability of ribonuclease A and ribonuclease T1 to digest single-stranded RNA, but not perfectly base-paired double-stranded RNA. In this respect RNA:RNA hybrids are more resistant to ribonuclease than RNA:DNA hybrids are to S1 nuclease, resulting in fewer artifacts. RNase protection has a number of advantages over Northern analysis in the quantitation of mRNA levels. First, the hybridization of probe and target RNA takes place in a very small volume with very favorable renaturation kinetics (1 ). Second, poly(A)⁺ RNA is rarely required since 10 μg of RNA are sufficient for the detection of mRNA species present at l–5 copies/cell. Third, the RNase protection method allows the discrimination between closely homologous sequences, allowing the detection of specific mRNA species within a population of closely related sequences. Fourth, RNase protection is particularly suited to the quantitation mRNA species that are partially degraded (which is common with clinical samples) or too large to be found intact by Northern analysis, since the probe is generally significantly shorter than the target RNA.