Gangisetty Omkaram, Reddy Doodipala Samba
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M System Health Science Center, College Station, TX 77843-1114, USA.
J Neurosci Methods. 2009 Jun 30;181(1):58-66. doi: 10.1016/j.jneumeth.2009.04.016. Epub 2009 May 3.
The GABA-A receptor plays a critical role in inhibitory neurotransmission in the brain. Quantitation of GABA-A receptor subunits in various brain regions is essential to understand their role in plasticity and brain disorders. However, conventional RNA assays are tedious and less sensitive for use in studies of subunit plasticity. Here we describe optimization of a sensitive assay of GABA-A receptor subunit gene expression by TaqMan real-time PCR. For each subunit gene, a set of primers and TaqMan fluorogenic probe were designed to specifically amplify the target template. The TaqMan methodology was optimized for quantification of mouse GABA-A receptor subunits (alpha(1-6), beta(1-3), gamma(2), and delta) and GAPDH. The TaqMan reaction detected very low levels of gene expression ( approximately 100 template copies of cDNA). A standard curve for GAPDH and one of the target genes, constructed using the cDNA, revealed slopes around -3.4 (r(2)=0.990), reflecting similar optimum PCR efficiencies. The methodology was utilized for quantification of the GABA-A receptor alpha(4)-subunit, which is known to upregulate following withdrawal from chronic progesterone or neurosteroids. Our results show that the alpha(4)-subunit expression increased threefold in the hippocampus following neurosteroid withdrawal in mice. The TaqMan PCR assay allows sensitive, high-throughput transcriptional profiling of complete GABA-A receptor subunit family, and thus provides specific tool for studies of GABA-A receptor subunit plasticity in neurological and psychiatric animal models.
GABA-A受体在大脑的抑制性神经传递中起关键作用。定量分析不同脑区的GABA-A受体亚基对于理解它们在可塑性和脑部疾病中的作用至关重要。然而，传统的RNA检测方法繁琐，且在亚基可塑性研究中的敏感性较低。在此，我们描述了通过TaqMan实时PCR优化GABA-A受体亚基基因表达的敏感检测方法。针对每个亚基基因，设计了一组引物和TaqMan荧光探针以特异性扩增目标模板。对TaqMan方法进行了优化，用于定量小鼠GABA-A受体亚基（α(1 - 6)、β(1 - 3)、γ(2)和δ）以及甘油醛-3-磷酸脱氢酶（GAPDH）。TaqMan反应能检测到极低水平的基因表达（约100个cDNA模板拷贝）。使用cDNA构建的GAPDH和其中一个目标基因的标准曲线显示斜率约为 - 3.4（r(2)=0.990），反映出相似的最佳PCR效率。该方法用于定量GABA-A受体α(4)亚基，已知该亚基在从慢性孕酮或神经甾体撤药后会上调。我们的结果表明，在小鼠神经甾体撤药后，海马体中α(4)亚基的表达增加了三倍。TaqMan PCR检测方法能够对完整的GABA-A受体亚基家族进行敏感、高通量的转录谱分析，从而为神经和精神疾病动物模型中GABA-A受体亚基可塑性的研究提供了特定工具。
