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.
Original language | English (US) |
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Pages (from-to) | 75-83 |
Number of pages | 9 |
Journal | Biotechnic and Histochemistry |
Volume | 76 |
Issue number | 2 |
State | Published - 2001 |
Externally published | Yes |
Keywords
- Facial motor nucleus
- Frozen sections
- In situ RT-PCR
- mRNA
- Nonradioactive labeling
- PAC-R splice variants
ASJC Scopus subject areas
- Anatomy
- Applied Microbiology and Biotechnology