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Nuclease protection assays (NPAs), including both ribonuclease protection assays (RPAs) and S1 nuclease assays, are an extremely sensitive method for the detection, quantitation and mapping of specific RNAs in a complex mixture of total cellular RNA. The basis of NPAs is a solution hybridization of a single-stranded, discrete sized antisense probe(s) to an RNA sample (see Figure 1). The small volume solution hybridization is far more efficient than traditional membrane-based hybridization, and can accommodate up to 100 礸 of total or poly(A) RNA. After hybridization, any remaining unhybridized probe and sample RNA are removed by digestion with a mixture of nucleases. Then, in a single step reaction, the nucleases are inactivated and the remaining probe:target hybrids are precipitated. These products are separated on a denaturing polyacrylamide gel and are visualized by autoradiography. If nonisotopic probes are used, samples are visualized by transferring the gel to a membrane and performing secondary detection.

Figure 1. Detection of Specific mRNA Species Using a Nuclease Protection Assay. 

NPAs are the method of choice for the simultaneous detection of several RNA species. During solution hybridization and subsequent analysis, individual probe/target interactions are completely independent of one another. Thus, several RNA targets and internal controls can be assayed simultaneously (up to twelve have been used in the same reaction), provided that the protected fragment of individual probes are of different lengths. NPAs are also commonly used to precisely map mRNA termini and intron/exon junctions.

RNA Quantitation

To quantitate mRNA levels using NPAs, the intensities of probe fragments protected by the sample RNA are compared to the intensities generated from either an endogenous internal control (relative quantitation) or known amounts of sense strand RNA (absolute quantitation). For more information on using NPAs for quantitation, see Technical Bulletin 151, "Use of Internal and External Standards or Reference RNAs for Accurate Quantitation of RNA Levels."