Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.
Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.
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DOI：
10.1007/s10529-014-1652-9
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发表时间：
2015-01
影响因子：
2.7
通讯作者：
Wang, Yimin
中科院分区：
文献类型：
--
作者：
Li, Xiuying;Yang, Qiwei;Bai, Jinping;Xuan, Yali;Wang, Yimin
参考基因标准化是定量逆转录 PCR (RT-qPCR) 分析的首选方法。内参基因的稳定性对于准确的实验结果和结论至关重要。我们评估了四种不同人类间充质干细胞 (MSC) 中发现的八种常用参考基因的表达稳定性。使用 geNorm、NormFinder 和 BestKeeper 算法，我们表明 β-2-微球蛋白和肽基脯氨酰异构酶 A 是标准化从 MSC 获得的 RT-qPCR 数据的最佳参考基因，而 TATA 盒结合蛋白由于其表达的广泛变异性而不适合。我们的研究结果强调了验证 qPCR 分析参考基因的重要性。我们提供了用于标准化的参考基因的简短列表，并推荐了一些市售软件程序作为验证参考基因的快速方法。我们还证明，经常使用的两个参考基因，β-肌动蛋白和甘油醛-3-磷酸脱氢酶，在许多情况下并不总是成功。本文的在线版本 (doi:10.1007/s10529-014-1652-9) 包含补充材料，可供授权用户使用。
Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases. The online version of this article (doi:10.1007/s10529-014-1652-9) contains supplementary material, which is available to authorized users.
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影响因子：
5.4
作者：
Li X;Bai J;Ji X;Li R;Xuan Y;Wang Y
通讯作者：
Wang Y
影响因子：
2.1
作者：
Manochantr, S.;U-Pratya, Y.;Issaragrisil, S.
通讯作者：
Issaragrisil, S.
影响因子：
4.3
作者：
Raicevic, Gordana;Najar, Mehdi;Lagneaux, Laurence
通讯作者：
Lagneaux, Laurence
影响因子：
5.2
作者：
Kern, Susanne;Eichler, Hermann;Bieback, Karen
通讯作者：
Bieback, Karen
影响因子：
4.3
作者：
Najar, Mehdi;Raicevic, Gordana;Lagneaux, Laurence
通讯作者：
Lagneaux, Laurence