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首页> 外文期刊>Journal of Neuroscience Methods >The optimization of TaqMan real-time RT-PCR assay for transcriptional profiling of GABA-A receptor subunit plasticity.
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The optimization of TaqMan real-time RT-PCR assay for transcriptional profiling of GABA-A receptor subunit plasticity.

机译：TaqMan实时RT-PCR测定法对GABA-A受体亚基可塑性的转录谱分析的优化。

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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.
机译：GABA-A受体在抑制大脑中的神经传递中起关键作用。量化各个大脑区域中的GABA-A受体亚基，对于了解它们在可塑性和脑部疾病中的作用至关重要。然而，常规的RNA测定用于亚基可塑性的研究是乏味的并且敏感性较低。在这里，我们描述了TaqMan实时PCR对GABA-A受体亚基基因表达敏感测定的优化。对于每个亚基基因，设计了一组引物和TaqMan荧光探针以特异性扩增靶模板。 TaqMan方法已针对小鼠GABA-A受体亚基（α（1-6），β（1-3），γ（2）和δ）和GAPDH的定量进行了优化。 TaqMan反应检测到非常低的基因表达水平（大约100个cDNA模板拷贝）。 GAPDH和使用cDNA构建的靶基因之一的标准曲线显示-3.4附近的斜率（r（2）= 0.990），反映出相似的最佳PCR效率。该方法用于量化GABA-A受体alpha（4）-亚基，已知该亚基从慢性孕激素或神经甾体戒断后会上调。我们的结果表明，在小鼠中神经甾体戒断后，海马中的alpha（4）-亚基表达增加了三倍。 TaqMan PCR测定法可对完整的GABA-A受体亚基家族进行灵敏的高通量转录分析，从而为神经和精神病动物模型中GABA-A受体亚基可塑性的研究提供了特定的工具。

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