Mogal Ashish, Abdulkadir Sarki A
Department of Pathology, Vanderbilt University, School of Medicine, Nashville, TN 37232, USA.
Mol Cell Probes. 2006 Apr;20(2):81-6. doi: 10.1016/j.mcp.2005.09.008. Epub 2005 Dec 2.
In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.
在定量逆转录聚合酶链反应（qRT-PCR）中，基因表达分析依赖于使用看家基因（如18S rRNA、甘油醛-3-磷酸脱氢酶（GAPDH）和β-肌动蛋白）进行标准化。然而，据报道，它们的表达变异性是由药物治疗、病理状态和细胞周期阶段等因素引起的。癌症研究的一个新兴领域专注于确定表观遗传改变（如组蛋白修饰和DNA甲基化）在癌症发生和发展中的作用。组蛋白乙酰化是目前研究最多的修饰，并且已经通过使用组蛋白去乙酰化酶抑制剂（HDACi）进行了探究。此外，目前正在探索调节组蛋白乙酰化作为癌症治疗的一种策略，并且HDACi在抑制肿瘤发生和转移方面已显示出前景。曲古抑菌素A（TSA）是使用最广泛的HDACi。因此，我们致力于确定TSA处理后用于RT-PCR的合适内参。我们对经TSA处理的小鼠前列腺组织外植体、人前列腺癌（LNCaP）细胞和人乳腺癌（T-47D和ZR-75-1）细胞进行了定量RT-PCR分析。比较了在溶剂对照与TSA处理样品中包括18S rRNA、β-肌动蛋白、GAPDH和核糖体高碱性23 kDa蛋白（rb 23-kDa，RPL13A）在内的看家基因的表达。我们的结果显示，在TSA处理后的小鼠前列腺外植体和人前列腺癌细胞系（LNCaP）中，18S rRNA、β-肌动蛋白mRNA和GAPDH mRNA水平存在显著差异。此外，在两个人乳腺癌细胞系（T-47D和ZR-75-1）中，18S rRNA、β-肌动蛋白mRNA和GAPDH mRNA水平也有显著变化。然而，在所有测试条件下，RPL13A mRNA水平保持恒定。因此，我们建议在TSA处理期间使用RPL13A作为标准化的标准。
