title: Application and modification of in situ RT-PCR for detection and cellular localization of PAC1-R splice variant mRnas in frozen brain sections.
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.
authors: Department of Biology, Waseda University School of Education, Tokyo, Japan.
authors: C J
authors: CJ
authors: Zhou
authors: S
authors: S
authors: Kikuyama
authors: S
authors: S
authors: Shioda
mesh_terms: D000818:Animals; D001921:Brain / Q000033:anatomy & histology*; D001923:Brain Chemistry / Q000235:genetics*; D017931:DNA Primers; D003851:Deoxyribonucleases / Q000378:metabolism; D005557:Formaldehyde; D005615:Freezing; D007202:Indicators and Reagents; D009479:Neuropeptides / Q000378:metabolism; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation
chemical_list: C495403:Adcyap1 protein, rat; C495505:Adcyap1r1 protein, rat; D017931:DNA Primers; D007202:Indicators and Reagents; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D005557:Formaldehyde; D003851:Deoxyribonucleases