SYNTHESIS OF DIGOXIGENIN-LABELED CRNA PROBES FOR NONISOTOPIC INSITU HYBRIDIZATION USING REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

Nonisotopic methods of mRNA in situ hybridization have distinct advant ages over isotopic techniques. Nonisotopically labeled probes are stab le and nontoxic, have short detection times, demonstrate excellent spa tial resolution of their signals and have sensitivities comparable to radiolabeled probes. We developed a simple method of generating noniso topically labeled cRNA probes which is based on the reverse transcript ion polymerase chain reaction (RT-PCR) and used it to synthesize a pan el of probes for various murine extracellular matrix genes. Engelbreth -Holm-Swarm (EHS) tumor RNA was reverse transcribed and PCR was used t o amplify defined regions of multiple extracellular matrix protein gen es from the resulting first strand cDNAs. Bacteriophage promoters whic h had been incorporated into the PCR products were then used to genera te digoxigenin-conjugated antisense and sense cRNAs. The antisense pro bes were employed to detect the specific extracellular matrix protein mRNAs in the EHS tumor by in situ hybridization. This technique provid es a rapid and efficient alternative to conventional transcription sys tems which use plasmid vectors for the synthesis of digoxigenin-labele d cRNA probes.