Pohjanvirta Raimo, Niittynen Marjo, Lindén Jere, Boutros Paul C, Moffat Ivy D, Okey Allan B
Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
Chem Biol Interact. 2006 Mar 25;160(2):134-49. doi: 10.1016/j.cbi.2006.01.001. Epub 2006 Feb 8.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is an extremely sensitive, convenient and rapid method to measure mRNA levels in cells and tissues, and is gaining popularity in toxicology. To correct for sample-to-sample variation, normalization of the expression data is required. The conventional way to perform normalization is to select a reference gene whose expression is believed to remain stable across all experimental conditions, then relate the concentrations of gene(s) of interest to those of this housekeeping gene. Since recent evidence shows that some housekeeping genes are actually not as refractory to experimental manipulations as previously thought, we validated a large number (18) of commonly used housekeeping genes for acute toxicity studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an extremely potent environmental toxin known to regulate a wide variety of genes. Microarray and qRT-PCR analyses coherently demonstrated that about 50% of the housekeeping genes examined were responsive to TCDD in rat liver with the magnitudes of change up to nearly 10-fold. Extension of the study to spleen and hypothalamus verified that phosphoglycerate kinase 1 (Pgk1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) retained their basal expression levels in all experimental settings, although body weight loss-generated repression may mask a slight induction of GAPDH by TCDD in liver. These findings show that normalization genes for qRT-PCR must be carefully validated in advance, especially if the study involves a potent modifier of gene expression.
定量逆转录-聚合酶链反应（qRT-PCR）是一种极其灵敏、便捷且快速的方法，用于测量细胞和组织中的mRNA水平，在毒理学领域正变得越来越流行。为校正样本间的差异，需要对表达数据进行标准化。传统的标准化方法是选择一个被认为在所有实验条件下表达都保持稳定的管家基因，然后将感兴趣基因的浓度与该管家基因的浓度相关联。由于最近的证据表明，一些管家基因实际上并不像以前认为的那样对实验操作具有抗性，我们针对2,3,7,8-四氯二苯并-p-二恶英（TCDD）的急性毒性研究验证了大量（18个）常用的管家基因，TCDD是一种极强的环境毒素，已知可调节多种基因。微阵列和qRT-PCR分析一致表明，在所检测的管家基因中，约50%在大鼠肝脏中对TCDD有反应，变化幅度高达近10倍。将研究扩展到脾脏和下丘脑证实，磷酸甘油酸激酶1（Pgk1）和甘油醛-3-磷酸脱氢酶（GAPDH）在所有实验条件下都保持其基础表达水平，尽管体重减轻导致的抑制可能掩盖了TCDD在肝脏中对GAPDH的轻微诱导。这些发现表明，用于qRT-PCR的标准化基因必须预先仔细验证，特别是如果该研究涉及基因表达的强效调节剂。
