Validation of endogenous controls for quantitative gene expression analysis:: Application on brain cortices of human chronic alcoholics
Validation of endogenous controls for quantitative gene expression analysis:: Application on brain cortices of human chronic alcoholics
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DOI：
10.1016/j.brainres.2006.11.026
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发表时间：
2007-02-09
期刊：
影响因子：
2.9
通讯作者：
Ekstrom, Tomas J.
中科院分区：
文献类型：
--
作者：
Johansson, Sofia;Fuchs, Andrea;Ekstrom, Tomas J.
实时PCR由于其方法学灵敏度和可重复性而经常用于基因表达定量。通过标准化至一个或多个参考基因（通常为β-肌动蛋白（ACTB）、甘油醛-3-磷酸脱氢酶（GAPD）或核糖体RNA（18 S））来定量基因表达。然而，不同的环境或病理条件也可能影响正常化基因的表达，这可能严重影响定量结果的解释。本研究评估了16个基因是否经常用作内源性对照的表达研究，可以作为这样的比较慢性酗酒者和对照组的人脑组织。分析了慢性饮酒对前额叶和运动皮层的不同影响。在酗酒者和对照受试者中表达没有差异或差异很小的参考基因被发现对每个区域都是特异性的：前额叶皮层的β-肌动蛋白（ACTB）和核糖体大PO（RPLPO），而运动皮层的importin 8（IPO 8）和RNA聚合酶II（POLR 2A）。在16个分析的基因中，有4个在酗酒者和对照组之间表现出显着差异：磷酸甘油酸激酶（PGK 1），次黄嘌呤磷酸核糖转移酶（HPRT 1）和肽基脯氨酰异构酶A（PPIA）在运动皮层和β-2-微球蛋白（B2 M）在前额叶皮层。我们的研究证明了在人脑组织的实时PCR分析之前验证内源性对照基因的重要性。处方药和非处方药，病理或环境条件沿着酒精滥用可能会差异影响参考基因的表达。(c)2006 Elsevier B. V.保留所有权利。
Real-time PCR is frequently used for gene expression quantification due to its methodological sensitivity and reproducibility. The gene expression is quantified by normalization to one or more reference genes, usually beta-actin (ACTB), glyceralclehyde-3-phosphate dehydrogenase (GAPD) or to ribosomal RNA (18S). However, different environmental or pathological conditions might also influence the expression of normalizing genes, which could severely skew the interpretation of quantitative results. This study evaluates whether 16 genes frequently used as endogenous controls in expression studies, can serve as such for comparison of human brain tissues of chronic alcoholics and control subjects. The prefrontal and motor cortices that are affected differently by chronic alcohol consumption were analyzed. The reference genes that have no or small differences in expression in alcoholics and control subjects, were found to be specific for each region: beta-actin (ACTB) and ribosomal large PO (RPLPO) for the prefrontal cortex while importin 8 (IPO8) and RNA polymerase II (POLR2A) for the motor cortex. Four out of sixteen analyzed genes demonstrated significant differences in expression between alcoholics and controls: phosphoglycerate kinase (PGK1), hypoxanthine phosphoribosyl transferase (HPRT1) and peptidylprolyl isomerase A (PPIA) in the motor cortex and beta-2-microglobulin (B2M) in the prefrontal cortex. Our study demonstrates the importance of validation of endogenous control genes prior to real-time PCR analysis of human brain tissues. Prescribed and non-prescribed drugs, pathological or environmental conditions along with alcohol abuse may differentially influence expression of reference genes. (c) 2006 Elsevier B.V. All rights reserved.