Detection of RNAs by 3'-end labeling and RNase H digestion

This protocol describes an extremely sensitive procedure for detecting the presence of known or unknown RNAs in a complex mixture. A selectively enriched population of RNAs is subjected to 3'-end labeling with [(32)P]pCp, and labeled products are separated from unincorporated label. The labeled RNAs are hybridized to sequence-specific complementary oligodeoxynucleotides, treated with RNase H (which cleaves RNA in an RNA-DNA hybrid) and the products analyzed by electrophoresis through denaturing polyacrylamide gels with the appropriate controls. If the RNA of interest was present and hybridized to its complementary oligonucleotide, its digestion with RNase H will result in a shift in its mobility through the gel or, if the RNA was fully degraded, its band will not appear. If the RNA of interest is not cleaved in the presence of any known complementary oligodeoxynucleotides, then its position in the gel will remain unaltered. This result may suggest the presence of a new or unknown RNA that may be identified using a variety of cloning techniques or by direct chemical sequencing methods.