Normalization of gene expression using SYBR green qPCR: A case for paraoxonase 1 and 2 in Alzheimer's disease brains
Normalization of gene expression using SYBR green qPCR: A case for paraoxonase 1 and 2 in Alzheimer's disease brains
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DOI：
10.1016/j.jneumeth.2011.05.026
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发表时间：
2011-08-30
影响因子：
3
通讯作者：
Poirier, Judes
中科院分区：
文献类型：
--
作者：
Leduc, Valerie;Legault, Veronique;Poirier, Judes
验证参考基因的表达稳定性对于实时定量聚合酶链式反应(QPCR)数据的可靠标准化至关重要，但在人脑组织中研究这一问题的研究相对较少。因此，本研究的目的是在人类死后脑组织中寻找一组合适的内源性参考基因(ERG)，用于分析与阿尔茨海默病(AD)相关的潜在候选基因。应用SYBR Green技术检测了尸检证实的AD和非痴呆对照组(n=20)额叶皮质中10种常见ERG(ACTB、GAPDH、GPS1、GUSB、M-RIP、PGK1、POL2RF、PPIA、UBE2D2和YES1)的mRNA水平。然后，使用三个软件应用程序：geNorm、NormFinder和Best-Keeper，根据表达稳定性对这些水平进行排名。而PPIA和UBE2D2是表达最可靠的ERG。ACTS是最糟糕的。随后，使用PPIA和UBE2D2作为标准化ERGs，对AD组和对照组(n=80)额叶皮质对氧磷酶1(PON1)和对氧磷酶2(PON2)的mRNA水平进行定量，并用REST 2009程序进行分析。我们的结果表明，这两种对氧磷酶在人类额叶皮质中都有表达，并且与非痴呆对照组相比，阿尔茨海默病患者PON2而不是PON1的mRNA水平上调。然而，ANCOVA对结果的重新分析表明，AD组和对照组之间差异的显著性取决于用于标准化的ERG。因此，建议在分析数据时使用一种计算方法，以便纳入可能的混杂因素。(C)2011年，由爱思唯尔出版。
Validating the expression stability of reference genes is crucial for reliable normalization of real-time quantitative PCR (qPCR) data, but relatively few studies have investigated this issue in brain human tissues. The present study thus aimed at identifying in human post-mortem brain tissues a set of suitable endogenous reference genes (ERG) for the expression analysis of potential candidate genes associated with Alzheimer's disease (AD). The mRNA levels of ten common ERGs (ACTB, GAPDH, GPS1,GUSB,M-RIP, PGK1, POL2RF, PPIA, UBE2D2, and YES1) were determined in the frontal cortex of autopsy-confirmed AD and non-demented control cases (n = 20) using SYBR Green technology. Then, these levels were ranked according to their expression stability using three software applications: geNorm, NormFinder and Best-Keeper. Whereas PPIA and UBE2D2 were among the ERGs with the most reliable expression. ACTS was the worst. Subsequently, using PPIA and UBE2D2 as ERGs for normalization, the mRNA levels of paraoxonase 1 (PON1) and paraoxonase 2 (PON2) were quantified in the frontal cortex of AD and control cases (n = 80) and analyzed using the REST 2009 program. Our results indicate that both paraoxonases are expressed in the human frontal cortex and that PON2 but not PON1 mRNA levels are up-regulated in AD relative to non-demented controls. However, re-analysis of the results by ANCOVA indicated that the significance of the difference between AD and control groups depended upon the ERG used for normalization. The use of a computational method allowing the inclusion of possible confounding factors is thus recommended for the analysis of data. (C) 2011 Published by Elsevier B.V.