Gene expression analysis by real-time PCR: Experimental demonstration of PCR detection limits
Gene expression analysis by real-time PCR: Experimental demonstration of PCR detection limits
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DOI：
10.1016/j.ab.2012.09.029
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发表时间：
2013-01-15
影响因子：
2.9
通讯作者：
Albano, Rodolpho Mattos
中科院分区：
文献类型：
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作者：
Bernardo, Vagner;Ribeiro Pinto, Luis Felipe;Albano, Rodolpho Mattos
逆转录实时荧光定量聚合酶链反应（RT - qPCR）是定量基因表达的金标准。然而，由于聚合酶链反应的检测限（理论上为三个模板拷贝），对表现出较大表达变异性的基因进行定量具有挑战性。我们利用大鼠组织中高表达至低表达的基因，通过实验证明了这一限制，并发现它仅适用于描述存在随机事件和蒙特卡罗效应的反应。我们还确定了应使用的RNA输入下限，以防止人为的模板定量，并提出了一种在任何qPCR平台上评估RT - qPCR检测限的方法。（C）2012爱思唯尔公司。保留所有权利。
Reverse transcription followed by real-time PCR (RT-qPCR) is the gold standard for quantifying gene expression. However, because of PCR detection limits, theorized to be three template copies, the quantification of genes exhibiting great expression variability is challenging. Using genes with high to low expression in rat tissues we experimentally demonstrated this limit and found it to be applicable only for describing reactions in which stochastic events and the Monte Carlo effect are present. We also determined the lower limits of RNA input that should be used to prevent artifactual template quantification and we propose a methodology to assess RT-qPCR detection limits in any qPCR platform. (C) 2012 Elsevier Inc. All rights reserved.