Guideline to reference gene selection for quantitative real-time PCR
Guideline to reference gene selection for quantitative real-time PCR
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DOI：
10.1016/j.bbrc.2003.11.177
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发表时间：
2004-01-23
影响因子：
3.1
通讯作者：
Nitsche, A
中科院分区：
文献类型：
--
作者：
Radonic, A;Thulke, S;Nitsche, A
目前，实时定量聚合酶链式反应是快速、可靠地定量检测基因转录的首选方法。然而，为了准确比较不同样本或组织中的mRNA转录，选择合适的参考基因是至关重要的。最近，3-磷酸甘油醛脱氢酶和P-肌动蛋白被用于这一目的。然而，据报道，这些基因以及替代基因，如rRNA基因，不是合适的参考基因，因为它们的转录在各种实验环境中受到显著调控，并且在不同的组织中是可变的。因此，本研究采用实时定量聚合酶链式反应技术，比较了13个参考基因在16种不同组织以及12-O-十四酰佛波醇-13-乙酸酯和离子霉素刺激的CCRF-HSB-2细胞中的转录水平。结果表明，经典参考基因确实不适用，而RNA聚合酶II基因是CCRF-HSB-2细胞中在不同组织和刺激后表达最恒定的基因。(C)2003 Elsevier Inc.保留所有权利。
Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and P-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that "Classical" reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells. (C) 2003 Elsevier Inc. All rights reserved.