Coverslip mounted-immersion cycled in situ RT-PCR for the localization of mRNA in tissue sections

An improved method was developed for in situ reverse transcription-polymerase chain reaction (RT-PCR) to detect and localize mRNA in tissue sections. The coverslip mounted-immersion cycled (COSMIC) in situ RT-PCR technique combines the advantages of solution-phase PCR with the tissue immobilization necessary for in situ analysis. The tissue specimen is mounted on an AES-silane-coated coverslip, excess glass is removed and the sample is immersed in reaction mixture in a PCR tube and subjected to thermal cycling. Processing the section on the coverslip is efficient, the thin glass withstands the high temperature cycling and the tissue adheres securely through the process. The specimen is fully exposed to the reagents, and is heated uniformly and accurately according to temperatures programmed into the thermal cycler. An application is described for the detection and localization of the mRNA for surfactant protein A (SP-A) in fetal rat lung tissue.