Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury
Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury
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DOI：
10.1016/j.ab.2010.07.021
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发表时间：
2010-11-15
影响因子：
2.9
通讯作者：
Welham, Nathan V.
中科院分区：
文献类型：
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作者：
Chang, Zhen;Ling, Changying;Welham, Nathan V.
在基因芯片和实时定量聚合酶链式反应(qRT-PCR)平台中，对稳定表达的参考基因进行归一化相对定量是一种广泛使用的数据分析方法；然而，最近的证据表明，许多常用的参考基因在某些实验系统和情况下是不稳定的。因此，这项研究的主要目的是筛选和鉴定在声带粘膜损伤大鼠模型中稳定表达的参考基因。我们选择了9个候选参考基因，并对其表达稳定性进行了评价。Ablim1、Sptbn1和Wrnip1在模型特定的微阵列数据集中稳定表达，并通过2-(Delta CT)和基于配对比较(GeNorm)的分析在独立的qRT-PCR实验中进一步验证为合适的参考基因。对六个常用参考基因的平行分析确定Sdha是该组中唯一稳定表达的候选基因。老天啊。Sptbn1，以及Sdha和Sptbn1的几何平均值分别提供了目标基因Tgfb1的准确归一化；GAPDH，我们数据集中最不稳定的候选基因，提供了不准确的归一化和无效的实验结果。这些稳定的参考基因适用于声带黏膜损伤实验中靶基因表达的准确标准化。(C)2010 Elsevier Inc.保留所有权利。
Relative quantification by normalization against a stably expressed reference gene is a widely used data analysis method in microarray and quantitative real-time polymerase chain reaction (qRT-PCR) platforms; however, recent evidence suggests that many commonly utilized reference genes are unstable in certain experimental systems and situations. The primary aim of this study, therefore, was to screen and identify stably expressed reference genes in a well-established rat model of vocal fold mucosal injury. We selected and evaluated the expression stability of nine candidate reference genes. Ablim1, Sptbn1, and Wrnip1 were identified as stably expressed in a model-specific microarray dataset and were further validated as suitable reference genes in an independent qRT-PCR experiment using 2-(Delta CT) and pairwise comparison-based (geNorm) analyses. Parallel analysis of six commonly used reference genes identified Sdha as the only stably expressed candidate in this group. Sdha. Sptbn1, and the geometric mean of Sdha and Sptbn1 each provided accurate normalization of target gene Tgfb1; Gapdh, the least stable candidate gene in our dataset, provided inaccurate normalization and an invalid experimental result. The stable reference genes identified here are suitable for accurate normalization of target gene expression in vocal fold mucosal injury experiments. (C) 2010 Elsevier Inc. All rights reserved.