IFISE, CONICET-UNR, Rosario, Argentina.
PLoS One. 2012;7(5):e36323. doi: 10.1371/journal.pone.0036323. Epub 2012 May 1.
Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.
肝毒性与外源性物质暴露引起的肝脏基因表达的重大变化有关。了解其潜在机制对于其临床诊断和治疗至关重要。microRNAs 是基因表达的关键调节因子，可控制 mRNA 的稳定性和翻译，在正常发育和病理过程中都是如此。测量基因转录本水平的经典技术是实时 qPCR，该技术已成功修改为确定 microRNAs 的水平。然而，在像 RT-qPCR 这样的多步骤方法中，为了获得准确的数据，必须使用内源性、稳定表达的参照基因进行标准化。由于候选参照基因的表达稳定性因实验因素而异，因此我们研究的目的是确定一组基因，以最佳地对急性肝毒性实验模型中的 microRNA 和 mRNA qPCR 表达数据进行标准化。大鼠用四种传统肝毒素：对乙酰氨基酚、四氯化碳、D-半乳糖胺和硫代乙酰胺处理，然后通过 RT-qPCR 测定两组候选参照基因的肝表达水平，一组用于 microRNA 标准化，另一组用于 mRNA 标准化，符合 MIQE 指南。在本研究中，我们报告称，传统的参照基因，如 U6 剪接体 RNA、β肌动蛋白和甘油醛-3-磷酸脱氢酶，在响应经典肝毒素时改变了它们的表达，因此不能在肝毒性研究中用作参照基因。使用 geNorm、NormFinder 和 BestKeeper 软件包，仅考虑那些未改变表达的候选参照基因，确定候选参照基因的稳定性排名。然后，进一步测试稳定的潜在候选基因，以找到可准确确定 mRNA 和 miRNA 水平的最佳参照基因组合。最后，将 MicroRNA-16/5S 核糖体 RNA 和 Beta 2 微球蛋白/18S 核糖体 RNA 的组合分别验证为急性肝毒性大鼠模型中 microRNA 和 mRNA 定量的最佳参照基因。
