Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition
Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition
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DOI：
10.1159/000090070
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发表时间：
2006-01-01
影响因子：
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通讯作者：
Turman, MA
中科院分区：
文献类型：
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作者：
Elberg, G;Elberg, D;Turman, MA
背景/目标：进行性肾纤维化疾病伴随着肌成纤维细胞的大量积累，如通过α平滑肌肌动蛋白（α SMA）表达所定义的。我们使用实时RT-PCR分析在原代培养的人肾小管细胞（RTC）转化为肌成纤维细胞的过程中，用转化生长因子β（1）（TGF-β 1）处理后，定量基因表达。我们在此报告mRNA定量常用的参考基因的局限性。研究方法：我们使用三种常见的内部对照（甘油醛-3-磷酸脱氢酶（GAPDH）、亲环素A和18 S rRNA），通过定量实时PCR测定了α SMA和巨核细胞白血病-1（MKL 1）（α SMA的转录调节因子）的表达。结果如下：GAPDH mRNA和亲环素A mRNA的表达，以及在较小程度上，18 S rRNA水平随培养时间和暴露于TGF-β 1而变化。因此，根据使用的参考基因，TGF-β 1似乎对MKL 1和α SMA的表达具有不同的影响。结论：原代培养的RTC转化为肌成纤维细胞是研究人类肾纤维化的一个有价值的系统。然而，TGF-β（1）治疗的参比基因表达的变异性表明，需要验证多个参比基因的mRNA定量，以在缺乏mRNA表达的通用内标物的情况下准确解释纤维化研究。版权所有（c）2006 S. Karger AG，巴塞尔。
Background/Aims: Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alpha SMA) expression. We quantitated gene expression using realtime RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor beta(1) (TGF-beta 1). We report herein the limitations of commonly used reference genes for mRNA quantitation. Methods: We determined the expression of alpha SMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alpha SMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Results: Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta 1. Thus, depending on which reference gene was used, TGF-beta 1 appeared to have different effects on expression of MKL1 and alpha SMA. Conclusions: RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta(1) treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. Copyright (c) 2006 S. Karger AG, Basel.