Application and modification of in situ RT-PCR for detection and cellular localization of PAC(1)-R splice variant mRNAs in frozen brain sections

Many important biopolymers such as neurotransmitters, modulators, transport ers and receptors are expressed in discrete regions of the brain or other t issues, and they often occur at extremely low concentrations; therefore, a sensitive detection system is required to map their distribution. To study the precise distribution patterns of the splice variants of the PAC(1) rece ptor, which specifically binds pituitary adenylate cyclase-activating polyp eptide (PACAP) with affinity in the nano- or picomolar range, we have appli ed an in situ reverse transcription-polymerase chain reaction (RT-PCR) tech nique in frozen tissue sections. We describe here a modified protocol using a single rTth enzyme, which can synthesize cDNA from RNA, then PCR amplify ing it in a single reaction mixture by varying the times and temperatures o f a thermal cycler. The primer pairs were the same as those used in the sol ution phase RT-PCR that had been used to obtain the expected bands of the a mplified products previously. A nonradioactive labeling system with digoxig enin conjugated with peroxidase or fluorescence for signal detection was co mpared. The gene expression of two PAC(1)-R splice variants in the rat moto r nucleus is first reported here.