The ribonuclease protection assay (RPA) has emerged as an important methodology for the detection, mapping, and quantification of RNAs. In this assay, total or cytoplasmic RNAs are hybridized to a high-specific activity antisense radioactive RNA probe synthesized by in vitro transcription from the SP6 or T7 promoter of an appropriate linearized plasmid template by the bacteriophage SP6 or T7 polymerase, respectively. The RNA hybrids are subjected to RNAse digestion and the protected products are resolved by denaturing polyacrylamide gel electrophoresis to allow detection of specific RNA fragments by subsequent autoradiography. RPAs are highly sensitive, the probes can be specifically targeted, and, when performed in probe excess, are quantitative, making them the method of choice for many analyses of RNA processing events.