The RNase protection assay (RPA) is a sensitive and quantitative assay for the detection of specific RNAs. Because some probes require an optimal temperature for efficient hybridization to the target RNAs, preliminary experiments to determine the optimal temperatures have to be done empirically, a process that is laborious and time-consuming. In order to simplify this process, a new gradient temperature hybridization procedure has been developed with the use of a thermocycler. This procedure eliminates the need for preliminary experiments to determine the optimal temperature of hybridization for a given probe.