Xi Dong, Keeler Benjamin, Zhang Wentong, Houle John D, Gao Wen-Jun
Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, United States.
J Neurosci Methods. 2009 Jan 30;176(2):172-81. doi: 10.1016/j.jneumeth.2008.09.013. Epub 2008 Sep 19.
The selective involvement of a subset of neurons in many psychiatric disorders, such as gamma-aminobutyric acid (GABA)-ergic interneurons in schizophrenia, creates a significant need for in-depth analysis of these cells. Here we introduce a combination of techniques to examine the relative gene expression of N-methyl-d-aspartic acid (NMDA) receptor subtypes in GABAergic interneurons from the rat prefrontal cortex. Neurons were identified by immunostaining, isolated by laser microdissection and RNA was prepared for reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. These experimental procedures have been described individually; however, we found that this combination of techniques is powerful for the analysis of gene expression in individual identified neurons. This approach provides the means to analyze relevant molecular mechanisms that are involved in the neuropathological process of a devastating brain disorder.
许多精神疾病中都存在一部分神经元的选择性受累,比如精神分裂症中γ-氨基丁酸(GABA)能中间神经元受累,这使得对这些细胞进行深入分析变得极为必要。在此,我们介绍一种技术组合,用于检测大鼠前额叶皮质GABA能中间神经元中N-甲基-D-天冬氨酸(NMDA)受体亚型的相对基因表达。通过免疫染色鉴定神经元,利用激光显微切割进行分离,然后制备RNA用于逆转录聚合酶链反应(RT-PCR)和实时PCR。这些实验步骤此前已分别进行过描述;然而,我们发现这种技术组合对于分析单个已鉴定神经元中的基因表达非常有效。这种方法为分析涉及一种毁灭性脑部疾病神经病理过程的相关分子机制提供了手段。