COVERSLIP MOUNTED-IMMERSION CYCLED IN-SITU RT-PCR FOR THE LOCALIZATION OF MESSENGER-RNA IN TISSUE-SECTIONS

An improved method was developed for in situ reverse transcription-pol ymerase chain reaction (RT-PCR) to detect and localize mRNA in tissue sections. The coverslip mounted-immersion cycled (COSMIC) in situ RT-P CR technique combines the advantages of solution-phase PCR with the ti ssue immobilization necessary for in situ analysis. The tissue specime n is mounted on an AES-silane-coated coverslip, excess glass is remove d and the sample is immersed in reaction mixture in a PCR tube and sub jected 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 accor ding to temperatures programmed into the thermal cycler. An applicatio n is described for the detection and localization of the mRNA for surf actant protein A (SP-A) in fetal rat lung tissue.