The RNase Protection Assay

A sensitive method for quantitation of mRNA in gene transfer studies is mRNA protection using end-labeled DNA probes. In addition, this technique also provides structural information about the transcript under study (1). In vitro labeled antisense RNAcan be used as an alternative to end-labeled DNA probes (2,3. RNA probes have attained wide popularity because of the ease of synthesis and high yield of probe in a labeling reaction. This has been made possible by the recent characterization of single subunit bacteriophage RNA polymerases and their promoters (4,5). Currently, three different bacteriophage RNA polymerase/promoter combinations are in use. They are derived from bacteriophage SP6 of Salmonella typhimurium (3), bacteriophage T3 of E. coli (6), and bacteriophage T7 of E. coli (6). These RNA polymerases are ideal for in vitro synthesis of labeled transcripts because of their ease of purification, stability, high rate of polymerization (10 times faster than E. coli), and their high specificity resulting from the recognition of large promoter sequences.