Jastrow Aaron, Gordon Denise A, Auger Kasie M, Punska Elizabeth C, Arcaro Kathleen F, Keteles Kristen, Winkelman Dana, Lattier David, Biales Adam, Lazorchak James M
Region 5 Laboratory, US Environmental Protection Agency, Chicago, Illinois, USA.
National Exposure Research Laboratory, US Environmental Protection Agency Office of Research and Development, Cincinnati, Ohio, USA.
Environ Toxicol Chem. 2017 Nov;36(11):3102-3107. doi: 10.1002/etc.3885. Epub 2017 Aug 14.
The egg yolk precursor protein vitellogenin is widely used as a biomarker of estrogen exposure in male fish. However, standardized methodology is lacking and little is known regarding the reproducibility of results among laboratories using different equipment, reagents, protocols, and data analysis programs. To address this data gap we tested the reproducibility across laboratories to evaluate vitellogenin gene (vtg) expression and assessed the value of using a freely available software data analysis program. Samples collected from studies of male fathead minnows (Pimephales promelas) exposed to 17α-ethinylestradiol (EE2) and minnows exposed to processed wastewater effluent were evaluated for vtg expression in 4 laboratories. Our results indicate reasonable consistency among laboratories if the free software for expression analysis LinRegPCR is used, with 3 of 4 laboratories detecting vtg in fish exposed to 5 ng/L EE2 (n = 5). All 4 laboratories detected significantly increased vtg levels in 15 male fish exposed to wastewater effluent compared with 15 male fish held in a control stream. Finally, we were able to determine that the source of high interlaboratory variability from complementary deoxyribonucleic acid (cDNA) to quantitative polymerase chain reaction (qPCR) analyses was the expression analysis software unique to each real-time qPCR machine. We successfully eliminated the interlaboratory variability by reanalyzing raw fluorescence data with independent freeware, which yielded cycle thresholds and polymerase chain reaction (PCR) efficiencies that calculated results independently of proprietary software. Our results suggest that laboratories engaged in monitoring programs should validate their PCR protocols and analyze their gene expression data following the guidelines established in the present study for all gene expression biomarkers. Environ Toxicol Chem 2017;36:3102-3107. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.
卵黄前体蛋白卵黄原蛋白被广泛用作雄性鱼类雌激素暴露的生物标志物。然而，目前缺乏标准化方法，对于使用不同设备、试剂、方案和数据分析程序的实验室之间结果的可重复性了解甚少。为填补这一数据空白，我们测试了不同实验室间的可重复性，以评估卵黄原蛋白基因（vtg）的表达，并评估使用免费软件数据分析程序的价值。从暴露于17α-乙炔雌二醇（EE2）的雄性黑头呆鱼（Pimephales promelas）研究以及暴露于处理后废水排放的呆鱼研究中收集的样本，在4个实验室中评估了vtg的表达。我们的结果表明，如果使用用于表达分析的免费软件LinRegPCR，各实验室之间具有合理的一致性，4个实验室中有3个检测到暴露于5 ng/L EE2的鱼类中存在vtg（n = 5）。与15条饲养在对照溪流中的雄性呆鱼相比，所有4个实验室均检测到15条暴露于废水排放的雄性呆鱼中vtg水平显著升高。最后，我们能够确定，从互补脱氧核糖核酸（cDNA）到定量聚合酶链反应（qPCR）分析，实验室间高变异性的来源是每台实时qPCR机器特有的表达分析软件。通过使用独立的免费软件重新分析原始荧光数据，我们成功消除了实验室间的变异性，该软件产生的循环阈值和聚合酶链反应（PCR）效率与专有软件无关，可独立计算结果。我们的结果表明，参与监测计划的实验室应按照本研究为所有基因表达生物标志物制定的指南，验证其PCR方案并分析其基因表达数据。《环境毒理学与化学》2017年；36:3102 - 3107。2017年由Wiley Periodicals Inc.代表SETAC出版。本文是美国政府的作品，因此在美国属于公共领域。
