Quantitation of GABA transporter 3 (GAT3) mRNA in rat brain by competitive RT-PCR
Quantitation of GABA transporter 3 (GAT3) mRNA in rat brain by competitive RT-PCR
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DOI：
10.1016/s1385-299x(99)00039-2
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发表时间：
1999-12-01
期刊：
影响因子：
--
通讯作者：
Hiemke, C
中科院分区：
文献类型：
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作者：
Ficková, M;Dahmen, N;Hiemke, C
γ-氨基丁酸是大脑中主要的抑制性神经递质。GABA转运体(GATs)将GABA从突触间隙中移除。到目前为止，已经克隆了5个不同的GABA转运蛋白，分别命名为GAT1到GAT4和vGAT。为了研究与药物增强GABA能传递相关的耐受和依赖的机制，我们分析了不同条件下不同脑区GATS的mRNA表达水平。在这篇文章中，我们描述了我们的GAT3mRNA表达测量方法，并通过对照实验对各个步骤进行了验证。以竞争对手cRNA为内标，进行竞争性逆转录聚合酶链式反应(RT-PCR)。在从不同组织中提取的总RNA中加入不同量的竞争对手cRNA，进行逆转录和聚合酶链式反应。扩增产物为GAT野生型片段和竞争片段。聚合酶链式反应产物用凝胶电泳法分离，测定条带强度，回归分析计算GAT3mRNA的相对丰度和绝对丰度。我们实验室的验证实验表明，该方法的批内可变性为6%，批间可变性为15%。(C)1999 Elsevier Science B.V.保留所有权利。
Gamma-amino butyric acid is the major inhibitory neurotransmitter in the brain. GABA transporters (GATs) remove GABA from the synaptic cleft. Till now, five distinct GABA transporters have been cloned and termed consecutively GAT1 to GAT4 and vGAT. To study the mechanisms by which tolerance and dependence associated with drugs enhancing GABAergic transmission is brought upon we analysed the mRNA expression levels of GATs in various brain regions under different conditions. In this paper, we describe our protocol for measurement of GAT3 mRNA expression, and its validation through control experiments for the various steps. We performed competitive reverse transcription and polymerase chain reaction (RT-PCR) with a competitor cRNA as internal standard. Different amounts of competitor cRNA were added to total RNA prepared from different tissue samples, reverse-transcribed and PCR amplified. The PCR amplification gave two products: the GAT wild type fragment and the competitor fragment. PCR products were separated by gel electrophoresis and band intensities were determined from which the relative and absolute abundance of GAT3 mRNA was calculated by regression analysis. Validation experiments in our laboratory showed a 6% intra-assay and a 15% inter-assay variability of this method. (C) 1999 Elsevier Science B.V. All rights reserved.