Production of Labeled cRNA Probes without Vector Cloning: Application to Localization of Basic Fibroblast Growth Factor and Tyrosine Hydroxylase mRNAs by in Situ Hybridization Histochemistry

Abstract

We describe an approach for generating labeled, single-stranded cRNA probes, using the polymerase chain reaction with primers containing RNA polymerase promoter sequences. Transcription reactions using the amplified products and RNA polymerases yield, for the most part, full-length products. cRNA probes for basic fibroblast growth factor and tyrosine hydroxylase which have incorporated 35 S-labeled nucleotides were used successfully for in situ hybridization histochemistry. This method provides a significantly faster, less work-intensive procedure for obtaining labeled single-stranded RNA useful for nucleic acid hybridization studies.