Validation of housekeeping genes for normalizing RNA expression in real-time PCR
Validation of housekeeping genes for normalizing RNA expression in real-time PCR
复制标题
DOI：
10.2144/04371rr03
复制
发表时间：
2004-07-01
期刊：
影响因子：
2.7
通讯作者：
Zumla, A
中科院分区：
文献类型：
--
作者：
Dheda, K;Huggett, JF;Zumla, A
使用实时PCR等技术分析RNA表达，传统上使用参考或管家基因来控制样本之间的误差。这种做法正受到质疑，因为越来越清楚的是，某些管家基因可能在某些生物样本中有相当大的差异。我们使用实时反转录PCR （RT-PCR）来评估健康个体和结核病患者外周血单个核细胞培养物和全血中表达的13个管家基因的水平。管家基因是从常规使用的基因和据报道在人类T细胞培养中不变的基因中选择的。没有一种常用的内参基因（如甘油醛-磷酸脱氢酶（GAPDH））被发现适合作为内参，因为它们是高度可变的（最大变异性为30倍）。此外，先前发现的在人类T细胞培养中不变的基因在RNA表达方面也表现出很大的差异（最大变异的34倍）。在血液中不变的基因在外周血单核细胞培养中高度可变。我们的数据表明，指定人类酸性核糖体蛋白的RNA是人类肺结核mRNA水平正常化的最合适的管家基因。管家基因的验证对于特定的实验模型是高度特异性的，并且是评估任何新模型的关键组成部分。
Analysis of RNA expression using techniques like real-time PCR has traditionally used reference or housekeeping genes to control for error between samples. This practice is being questioned as it becomes increasingly clear that some housekeeping genes may vary considerably in certain biological samples. We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis. Housekeeping genes were selected from conventionally used ones and from genes reported to be invariant in human T cell culture. None of the commonly used housekeeping genes [e.g., glyceraldehyde-phosphate-dehydrogenase (GAPDH)] were found to be suitable as internal references, as they were highly variable (>30-fold maximal variability). Furthermore, genes previously found to be invariant in human T cell culture also showed large variation in RNA expression (>34-fold maximal variability). Genes that were invariant in blood were highly variable in peripheral blood mononuclear cell culture. Our data show that RNA specifying human acidic ribosomal protein was the most suitable housekeeping gene for normalizing mRNA levels in human pulmonary tuberculosis. Validations of housekeeping genes are highly specific for a particular experimental model and are a crucial component in assessing any new model.