Application of the polymerase chain reaction to the RNase protection assay for 5-HT(1B) receptor mRNA levels measurement in rat brain tissues.
The RNase protection assay (RPA) is an extremely sensitive procedure for detection of messenger RNA (mRNA) in complex sample mixture of total RNA. However, its usefulness has been limited by the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction (PCR) to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for the 5-hydroxytryptamine(1B) (5-HT(1B)) receptor. Radiolabeled riboprobe was then synthesized using the PCR product as a template and used in RPA to detect mRNA for 5-HT(1B) receptor in rat brain. The internal control was the beta-Actin mRNA. Due to the simplicity of its design and the lack of need for subcloning, the DNA template synthesis by PCR facilitates the implementation of the RPA. Since the 5-HT(1B) receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters and the protocol described here permits the determination of 5-HT(1B) receptor mRNA levels in the rat cerebellum, striatum, hippocampus and frontal cortex, this protocol is helpful in understanding the involvement of 5-HT(1B) receptors in various physiological phenomena.
Drogou
C
C
Département de Physiologie, IMASSA-CERMA, BP 73, 91223 Brétigny/Orge Cedex, France.
Chennaoui
M
M
Forestier
L
L
Grimaldi
B
B
Fillion
G
G
Guezennec
Y
Y
D000199:Actins / Q000235:genetics; D000818:Animals; D001923:Brain Chemistry; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin / Q000235:genetics*; D015203:Reproducibility of Results; D012260:Ribonucleases; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D000199:Actins; D012333:RNA, Messenger; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin; D012260:Ribonucleases