TOOLS TO MINIMIZE INTER‐LABORATORY VARIABILITY IN VITELLOGENIN GENE EXPRESSION MONITORING PROGRAMS
Environmental Toxicology & Chemistry, June 2017
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.
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 employing different data analysis programs. To address this data gap we tested the reproducibility across labs 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 four laboratories. Our results indicate reasonable consistency among laboratories if the free software for expression analysis, LinRegPCR, is used; with three out of four laboratories detecting vtg in fish exposed to 5 ng/L EE2 (n = 5). All four laboratories detected significantly increased vtg levels in 15 male fish exposed to wastewater effluent as compared to 15 male fish held in a control stream. Finally, we were able to determine that the source of high inter-laboratory variability from cDNA to qPCR analyses was the expression analysis software unique to each real-time quantitative polymerase chain reaction (qPCR) machine. We successfully eliminated the inter-laboratory variability by reanalyzing raw fluorescence data with independent freeware, which yielded cycle thresholds and PCR efficiencies that calculated results independently of proprietary software. Our results suggest that laboratories engaged in monitoring programs validate their PCR protocols and analyze their gene expression data following the guidelines established herein for all gene expression biomarkers. This article is protected by copyright. All rights reserved.
|Members of the public||3||75%|
|Readers by professional status||Count||As %|
|Student > Master||2||14%|
|Student > Ph. D. Student||2||14%|
|Readers by discipline||Count||As %|
|Agricultural and Biological Sciences||4||29%|
|Biochemistry, Genetics and Molecular Biology||3||21%|