Application and modification of in situ RT-PCR for detection and cellular localization of PAC1-R splice variant mRNAs in frozen brain sections

Author: Zhou CJ   Kikuyama S   Shioda S  

Publisher: Informa Healthcare

ISSN: 1473-7760

Source: Biotechnic & Histochemistry, Vol.76, Iss.2, 2001-03, pp. : 75-83

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Abstract

Many important biopolymers such as neurotransmitters, modulators, transporters and receptors are expressed in discrete regions of the brain or other tissues, 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 PAC1 receptor, which specifically binds pituitary adenylate cyclase-activating polypeptide (PACAP) with affinity in the nano- or picomolar range, we have applied an in situ reverse transcription-polymerase chain reaction (RT-PCR) technique in frozen tissue sections. We describe here a modified protocol using a single rTth enzyme, which can synthesize cDNA from RNA, then PCR amplifying it in a single reaction mixture by varying the times and temperatures of a thermal cycler. The primer pairs were the same as those used in the solution phase RT-PCR that had been used to obtain the expected bands of the amplified products previously. A nonradioactive labeling system with digoxigenin conjugated with peroxidase or fluorescence for signal detection was compared. The gene expression of two PAC1-R splice variants in the rat motor nucleus is first reported here.