Simultaneous determination of multiple transcripts and splice variants of a primary transcript using ribonuclease...

The ribonuclease (RNase) protection assay (RPA) is an extremely sensitive technique used to determine specific mRNAs from cell and tissue extracts. The present protocol presents detailed procedures for a conventional RPA using antisense RNA probes purified with a Fullengther apparatus. The Fullengther has the advantage of being a relatively quick and safe procedure compared to more conventional methods for purification of full-length RNA probes. Using this protocol, we sought to simultaneously determine multiple mRNA species, including splice variants of the type I receptor (PAC(1)) of pituitary adenylate cyclase-activating polypeptide (PACAP), an important mediator in the regulation of luteinizing hormone-releasing hormone (LHRH) synthesis by ovarian steroids such as progesterone [7]. PAC(1) has more than eight splice variants. We have been able to discriminate the hop1 variant from other splice variants. To improve our understanding of the regulation mechanism of genes that are related to each other, such as LHRH and PACAP, it is most important to simultaneously determine genes that are involved in the same physiological areas of regulation. Using only 5 microg of total RNA sample from a single rat preoptic area, we simultaneously determined five different transcripts, including four rare mRNA species such as LHRH, PACAP, and hop1 variant and other splice variants of PAC(1), as well as the internal control of cyclophilin mRNA. This protocol provides a method for the simultaneous determination of multiple transcripts using the RPA.