Application and modification of in situ RT-PCR for detection and cellular localization of PAC1-R splice variant mRnas in frozen brain sections.
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.
Zhou
C J
CJ
Department of Biology, Waseda University School of Education, Tokyo, Japan.
Kikuyama
S
S
Shioda
S
S
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
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