Horii Arata, Smith Paul F, Darlington Cynthia L
Vestibular Research Group, Department of Pharmacology and Toxicology, School of Medical Sciences, University of Otago, Dunedin, New Zealand.
Brain Res Brain Res Protoc. 2002 Feb;9(1):77-83. doi: 10.1016/s1385-299x(01)00139-8.
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.
逆转录聚合酶链反应（RT-PCR）是一种用于从少量组织中检测特定基因表达的强大工具，在灵敏度方面优于传统的RNA检测方法，如Northern印迹法和原位杂交（ISH）。然而，由于其指数性质，传统的RT-PCR不适合定量分析。最近，一种实时定量PCR方法已经被开发出来，以克服RT-PCR的缺点，例如定量分析。在这里，我们描述了使用荧光TaqMan探针的实时定量PCR，来研究包括前庭核复合体、下橄榄核和小脑绒球在内的中枢前庭系统中谷氨酸受体亚基/亚型基因（NR1、NR2A、GluR2、KA2、mGluR1、mGluR7）表达的区域差异。我们发现，实时定量PCR产生的结果与ISH等其他技术相似，但在相对速度和检测低水平基因表达的能力方面具有几个优势。我们认为，实时定量PCR是研究前庭脑干和小脑中其他神经递质受体基因表达的一种有用方法，并且预计在评估任何治疗后基因表达的变化时也会更加准确。
