The RNase protection assay is a sensitive method for transcription start-site localization. It begins with an RNA probe that is uniformly labeled by incorporation of one [α-32P]NTP, usually [α-32P]UTP. The RNA probe is synthesized by bacteriophage RNA polymerase (SP6, T7, or T3), which initiates transcription from specific phage promoters that have been engineered into a number of common plasmid vectors. The plasmid template contains a genomic DNA fragment spanning the region thought to contain the transcription start site for the gene of interest. This genomic fragment is subcloned into the plasmid downstream of the phage promoter in the antisense orientation, so that a portion of the 5′ end of the resulting RNA probe will be complementary to the mRNA of interest. The radiolabeled probe is annealed to cytoplasmic or total cellular mRNA purified from the cells of interest, with the hybridization reaction proceeding for several hours or overnight. RNase A and/or RNase T1 is then added to the hybridization reactions. These nucleases digest the single-stranded overhang regions of RNA molecules, but RNA–RNA hybrids are resistant to cleavage. This resistance forms the conceptual basis for the procedure; the region of the probe that anneals to the specific mRNA will be resistant to digestion. The length of the resistant region of the probe will correspond to the distance from the 5′ end of the probe to the transcription start site. The size of the resistant fragment can be determined by electrophoresis on a high-resolution, denaturing polyacrylamide gel.