Shekh Kamran, Tang Song, Niyogi Som, Hecker Markus
Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada; Toxicology Graduate Program, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada.
National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing, 100021, China.
Aquat Toxicol. 2017 Sep;190:217-227. doi: 10.1016/j.aquatox.2017.07.009. Epub 2017 Jul 24.
Gene expression analysis represents a powerful approach to characterize the specific mechanisms by which contaminants interact with organisms. One of the key considerations when conducting gene expression analyses using quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the selection of appropriate reference genes, which is often overlooked. Specifically, to reach meaningful conclusions when using relative quantification approaches, expression levels of reference genes must be highly stable and cannot vary as a function of experimental conditions. However, to date, information on the stability of commonly used reference genes across developmental stages, tissues and after exposure to contaminants such as metals is lacking for many vertebrate species including teleost fish. Therefore, in this study, we assessed the stability of expression of 8 reference gene candidates in the gills and skin of three different early life-stages of rainbow trout after acute exposure (24h) to two metals, cadmium (Cd) and copper (Cu) using qPCR. Candidate housekeeping genes were: beta actin (b-actin), DNA directed RNA polymerase II subunit I (DRP2), elongation factor-1 alpha (EF1a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein L8 (RPL8), and 18S ribosomal RNA (18S). Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative ΔCt method were employed to systematically evaluate the expression stability of these candidate genes under control and exposed conditions as well as across three different life-stages. Finally, stability of genes was ranked by taking geometric means of the ranks established by the different methods. Stability of reference genes was ranked in the following order (from lower to higher stability): HPRT<GAPDH<EF1a<G6PD<RPL8<DRP2<b-actin in gills of fish exposed to Cd; b-actin<GAPDH<G6PD<DRP2<RPL8<HPRT<EF1a in gills of fish exposed to Cu; RPL8<HPRT<GAPDH<G6PD<EF1a<DRP2<b-actin in the skin of fish exposed to Cd; and EF1a<GAPDH<RPL8<HPRT<G6PD<b-actin<DRP2 in the skin of fish exposed to Cu. The results demonstrated that the stability of reference genes depended on the metal, life-stage and/or organ in question. Thus, attention should be paid to these factors before selection of reference gene for relative quantification of the gene expressions.
基因表达分析是一种强大的方法，用于表征污染物与生物体相互作用的具体机制。在使用定量实时逆转录-聚合酶链反应（qPCR）进行基因表达分析时，一个关键的考虑因素是选择合适的参考基因，而这一点常常被忽视。具体而言，当使用相对定量方法得出有意义的结论时，参考基因的表达水平必须高度稳定，且不能随实验条件而变化。然而，迄今为止，对于包括硬骨鱼在内的许多脊椎动物物种，缺乏关于常用参考基因在发育阶段、组织以及暴露于金属等污染物后稳定性的信息。因此，在本研究中，我们使用qPCR评估了虹鳟鱼三个不同早期生命阶段的鳃和皮肤在急性暴露（24小时）于两种金属镉（Cd）和铜（Cu）后8个候选参考基因的表达稳定性。候选管家基因包括：β-肌动蛋白（b-actin）、DNA指导的RNA聚合酶II亚基I（DRP2）、延伸因子-1α（EF1a）、甘油醛-3-磷酸脱氢酶（GAPDH）、葡萄糖-6-磷酸脱氢酶（G6PD）、次黄嘌呤磷酸核糖转移酶（HPRT）、核糖体蛋白L8（RPL8）和18S核糖体RNA（18S）。采用geNorm、NormFinder、BestKeeper和比较ΔCt法四种算法，系统评估这些候选基因在对照和暴露条件下以及三个不同生命阶段的表达稳定性。最后，通过取不同方法确定的排名的几何平均值对基因稳定性进行排名。参考基因的稳定性按以下顺序排列（从稳定性较低到较高）：在暴露于Cd的鱼鳃中，HPRT＜GAPDH＜EF1a＜G6PD＜RPL8＜DRP2＜b-actin；在暴露于Cu的鱼鳃中，b-actin＜GAPDH＜G6PD＜DRP2＜RPL8＜HPRT＜EF1a；在暴露于Cd的鱼皮肤中，RPL8＜HPRT＜GAPDH＜G6PD＜EF1a＜DRP2＜b-actin；在暴露于Cu的鱼皮肤中，EF1a＜GAPDH＜RPL8＜HPRT＜G6PD＜b-actin＜DRP2。结果表明，参考基因的稳定性取决于所涉及的金属、生命阶段和/或器官。因此，在选择用于基因表达相对定量的参考基因之前，应注意这些因素。
