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) |
|---|---|
| Pages (from-to) | 75-83 |
| Number of pages | 9 |
| Journal | Biotechnic and Histochemistry |
| Volume | 76 |
| Issue number | 2 |
| State | Published - 2001 |
| Externally published | Yes |
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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
Cite this
Application and modification of in situ RT-PCR for detection and cellular localization of PAC1-R splice variant mRNAs in frozen brain sections. / Zhou, Chengji; Kikuyama, S.; Shioda, S.
In: Biotechnic and Histochemistry, Vol. 76, No. 2, 2001, p. 75-83.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Application and modification of in situ RT-PCR for detection and cellular localization of PAC1-R splice variant mRNAs in frozen brain sections
AU - Zhou, Chengji
AU - Kikuyama, S.
AU - Shioda, S.
PY - 2001
Y1 - 2001
N2 - 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.
AB - 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.
KW - Facial motor nucleus
KW - Frozen sections
KW - In situ RT-PCR
KW - mRNA
KW - Nonradioactive labeling
KW - PAC-R splice variants
UR - http://www.scopus.com/inward/record.url?scp=0035184454&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035184454&partnerID=8YFLogxK
M3 - Article
C2 - 11440308
AN - SCOPUS:0035184454
VL - 76
SP - 75
EP - 83
JO - Biotechnic and Histochemistry
JF - Biotechnic and Histochemistry
SN - 1052-0295
IS - 2
ER -