Department of Medicine, University of Alabama at Birmingham, 1900 University Blvd, Tinsley Harrison Tower 611J, Birmingham, AL 35294, USA.
Am J Nephrol. 2009;30(5):413-7. doi: 10.1159/000235993. Epub 2009 Sep 1.
Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use.
To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means.
Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes.
A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity.
内源性内部对照物（“参考”或“管家”基因）广泛应用于实时 PCR（RT-PCR）分析。它们的使用基于在研究的实验条件下稳定表达的前提。不幸的是，这些对照物中没有一种能在广泛的实验条件下满足这一前提；因此，不能推荐它们普遍使用。
为了确定哪些内源性 RT-PCR 对照物适合分析肾病改变的肾组织，我们在一个经过充分特征描述的囊性肾病模型中研究了 7 个轻度和 7 个重度受影响的整个肾脏组织中 16 种常用的“参考基因”的表达。通过 TaqMan RT-PCR 分析和 Affymetrix GeneChip 阵列确定这些 16 个基因的表达水平，进行归一化，并测试总体方差和均值的等效性。
两种统计方法以及基于 TaqMan 和 GeneChip 的方法都集中在排名前 4 的基因中的 3 个（Ppia、Gapdh 和 Pgk1）上，这些基因在研究的表型中具有最稳定的表达水平。
排名前几位的基因组合将为类似的研究提供一种合适的内源性内部对照物，适用于广泛的疾病严重程度的肾脏组织。
