Synthesis of digoxigenin-labeled cRNA probes for nonisotopic in situ hybridization using reverse transcription polymerase chain reaction.

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Nonisotopic methods of mRNA in situ hybridization have distinct advantages over isotopic techniques. Nonisotopically labeled probes are stable and nontoxic, have short detection times, demonstrate excellent spatial resolution of their signals and have sensitivities comparable to radiolabeled probes. We developed a simple method of generating nonisotopically labeled cRNA probes which is based on the reverse transcription polymerase chain reaction (RT-PCR) and used it to synthesize a panel of probes for various murine extracellular matrix genes. Engelbreth-Holm-Swarm (EHS) tumor RNA was reverse transcribed and PCR was used to amplify defined regions of multiple extracellular matrix protein genes from the resulting first strand cDNAs. Bacteriophage promoters which had been incorporated into the PCR products were then used to generate digoxigenin-conjugated antisense and sense cRNAs. The antisense probes were employed to detect the specific extracellular matrix protein mRNAs in the EHS tumor by in situ hybridization. This technique provides a rapid and efficient alternative to conventional transcription systems which use plasmid vectors for the synthesis of digoxigenin-labeled cRNA probes.