Approach for defining endogenous reference genes in gene expression experiments
Approach for defining endogenous reference genes in gene expression experiments
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DOI：
10.1016/j.ab.2004.02.037
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发表时间：
2004-06-15
影响因子：
2.9
通讯作者：
Van Dijk, W
中科院分区：
文献类型：
--
作者：
García-Vallejo, JJ;Van het Hof, B;Van Dijk, W
通过实时聚合酶链式反应 (PCR) 进行的基因表达定量彻底改变了基因表达分析领域。由于其敏感性和灵活性，它正在成为许多研究人员的首选方法。然而，仍然需要实施良好的标准化协议以促进数据交换和比较。我们为10个无关基因设计了引物，并开发了一个简单的方案来检测稳定表达的基因，这些基因适合用作内源参考基因，以进一步用于实时PCR获得的基因表达数据的标准化。使用该方案，我们能够将人蛋白体亚基 Y 鉴定为可靠的内源性参考基因，适用于用 TNFα、IL-4 或 IFNgamma 处理长达 18 小时的人脐静脉内皮细胞，以及从健康对照和患有 IgA 肾病的患者中分离的 B 细胞。可以考虑的其他可选内源参考基因是内皮细胞的磷酸甘露糖变位酶 (PPMM) 和肌动蛋白以及 B 细胞的 3-磷酸甘油醛脱氢酶和 PPMM。 (C) 2004 Elsevier Inc. 保留所有权利。
The quantification of gene expression by real-time polymerase chain reaction (PCR) has revolutionized the field of gene expression analysis. Due to its sensitivity and flexibility it is becoming the method of choice for many investigators. However, good normalization protocols still have to be implemented to facilitate data exchange and comparison. We have designed primers for 10 unrelated genes and developed a simple protocol to detect genes with stable expression that are suitable for use as endogenous reference genes for further use in the normalization of gene expression data obtained by real-time PCR. Using this protocol, we were able to identify human proteosome subunit Y as a reliable endogenous reference gene for human umbilical vein endothelial cells treated for up to 18 h with TNFalpha, IL-4, or IFNgamma and for B cells isolated from healthy controls and patients suffering from IgA nephropathy. Other optional endogenous reference genes that can be considered are phosphomannomutase (PPMM) and actin for endothelial cells and glyceraldehyde-3-phosphate dehydrogenase and PPMM for B cells. (C) 2004 Elsevier Inc. All rights reserved.