The Use of RNA Probes for the Analysis of Gene Expression: Northern Blot Hybridization and Ribonuclease Protection Assay

The isolation and characterization of RNA polymerases from the Salmonella phage SP6 and the E. coli phages T7 and T3 has revolutionized all aspects of the study of RNA metabolism (1 –6 ). Indeed, it is now possible to generate unlimited quantities of virtually any RNA molecule in a chemically pure form. This technology is based on a number of properties of the viral transcription units. First, and in contrast to their cellular counterparts, the enzymes are smgle-chain protems which were easily purified from phage-infected cells and are now produced by recombinant DNA technology. Second, they very specifically recognize their own promoters (7 and references therein), which are contiguous 17–20 bp long sequences rarely encountered in bacterial, plasmid or eukaryotic sequences. Third, the enzymes are highly processive, allowing the efficient synthesis of very long transcripts from DNA templates. In this chapter, the author will discuss the preparation of the DNA templates, the transcription from the templates of labeled synthetic RNA molecules, commonly called riboprobes, and their use in Northern and RNase protection assays.