Application of real-time quantitative polymerase chain reaction to quantification of glutamate receptor gene expression in the vestibular brainstem and cerebellum.
Reverse transcription-polymerase chain reaction (RT-PCR) is a powerful tool to detect specific gene expression from a small amount of tissue, which is superior to the traditional RNA assays such as Northern blotting and in situ hybridization (ISH) in terms of sensitivity. However, conventional RT-PCR is not suitable for quantification due to its exponential nature. Recently, a real-time quantitative PCR method has been developed to overcome the weak points of RT-PCR, e.g. quantification. Here we describe the use of real-time quantitative PCR using a fluorescent TaqMan probe, to study the regional differences in expression of glutamate receptor subunit/subtype genes (NR1, NR2A, GluR2, KA2, mGluR1, mGluR7) in the central vestibular system including the vestibular nucleus complex, inferior olive and cerebellar flocculus. We found that real-time quantitative PCR yielded similar results to other techniques such as ISH but offered several advantages in terms of relative speed and ability to detect low levels of gene expression. We suggest that real-time quantitative PCR is a useful method to study gene expression for other neurotransmitter receptors in the vestibular brainstem and cerebellum, and is also expected to be more accurate to assess the changes in gene expression following any treatment.
Horii
Arata
A
Vestibular Research Group, Department of Pharmacology and Toxicology, School of Medical Sciences, University of Otago, Dunedin, New Zealand. ahorii@ent.med.osaka-u.ac.jp
Smith
Paul F
PF
Darlington
Cynthia L
CL
D000818:Animals; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis; D005786:Gene Expression Regulation / Q000502:physiology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017470:Receptors, Glutamate / Q000235:genetics*; D013997:Time Factors; D014726:Vestibular Nuclei / Q000166:cytology / Q000378:metabolism*
D018076:DNA, Complementary; D012333:RNA, Messenger; D017470:Receptors, Glutamate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases