RNase Protection Assay
The RNase Protection Assay (RPA) is a specific, sensitive, and qualitative method for the detection, mapping, and quantitation of specific mRNAs.
The RPA is at least 10-fold more sensitive than Northern blot analysis and is more accurate and direct than RT-PCR analysis. In addition, RPA provides information that cannot be obtained reliably by other methods. For example, mapping transcription start sites, studying intron-exon junctions, and detecting very small differences in related transcripts can be achieved with RPA. The RNase protection assay can be performed with either total RNA or poly A+ RNA, and the results are not dependent upon having purified or non-degraded RNA.
At MDS a labeled, single-stranded antisense RNA probe is allowed to hybridize to the target RNA. RNA probe molecules and transcripts that do not form hybrids are degraded by a mixture of RNases. The final inactivation of RNases and the precipitation of protected RNA hybrids are performed simultaneously. Electrophoresis followed by autoradiography reveals the presence, size, and relative level of RNA that was protected by the antisense probe.