Analysis of Gene Expression by Multiprobe RNase Protection Assay

RNase protection assay (RPA) is becoming an increasingly popular method for the detection and quantitation of RNA levels in cells and tissues (1 –3 ). Hybridization is conducted in solution using an excess of a labeled antisense single-stranded RNA as probe. Thus, hybridization of the probe with target RNA results in the formation of stable, double-stranded RNA-RNA hybrids. After hybridization, the excess probe is removed by digestion with single-strand specific RNase, leaving behind only those probe molecules that were “protected” from digestion by virtue of having formed a duplex with their complementary mRNA target. These protected hybrids are denatured and separated from remaining labeled probe using standard sequencing polyacrylamide gel electrophoresis. The separated protected probe can then be visualized using routine autoradiography. In comparison with other RNA detection methods, such as Northern blot analysis or RT-PCR, the RPA has a number of advantages. These include:

1.	High sensitivity and specificity.
2.	Small sample requirement.
3.	Tolerant of RNA degradation.
4.	Easy quantitation.
5.	Rapid and simultaneous analysis of multiple target transcripts.
6.	High throughput analysis.
7.	Construction and use of “designer” probe sets.