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

Chengji Zhou, S. Kikuyama, S. Shioda

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)75-83
Number of pages9
JournalBiotechnic and Histochemistry
Volume76
Issue number2
StatePublished - 2001
Externally publishedYes

Fingerprint

Frozen Sections
Reverse Transcription
Polymerase Chain Reaction
Messenger RNA
Brain
Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I
Neurotransmitter Transport Proteins
Pituitary Adenylate Cyclase-Activating Polypeptide
Digoxigenin
Neurotransmitter Receptor
Biopolymers
Peroxidase
Complementary DNA
Hot Temperature
Fluorescence
RNA
Gene Expression
Temperature
Enzymes

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

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AU - Kikuyama, S.

AU - Shioda, S.

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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

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KW - Nonradioactive labeling

KW - PAC-R splice variants

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