title: DNA microarray application in ecotoxicology: experimental design, microarray scanning, and factors affecting transcriptional profiles in a small fish species.
abstract: The research presented here is part of a larger study of the molecular mode of action of endocrine-disrupting chemicals targeting the hypothalamic-pituitary-gonadal axis in zebrafish (Danio rerio). It addresses several issues critical to microarray application in aquatic ecotoxicology: experimental design, microarray scanning, gene expression intensity distribution, and the effect of experimental parameters on the zebrafish transcriptome. Expression profiles from various tissues of individual zebrafish exposed to 17alpha-ethinylestradiol (30 ng/L), fadrozole (25 micro.g/L), or 17beta-trenbolone (3.0 microg/L) for 48 or 96 h were examined with the Agilent Oligo Microarray (G2518A). As a flexible and efficient alternative to the designs commonly used in microarray studies, an unbalanced incomplete block design was found to be well suited for this work, as evidenced by high data reproducibility, low microarray-to-microarray variability, and little gene-specific dye bias. Random scanner noise had little effect on data reproducibility. A low-level, slightly variable Cyanine 3 (Cy3) contaminant was revealed by hyperspectral imaging, suggesting fluorescence contamination as a potential contributor to the large variance associated with weakly expressed genes. Expression intensities of zebrafish genes were skewed toward the lower end of their distribution range, and more weakly expressed genes tended to have larger variances. Tissue type, followed in descending order by gender, chemical treatment, and exposure duration, had the greatest effect on the overall gene expression profiles, a finding potentially critical to experimental design optimization. Overall, congruence was excellent between quantitative polymerase chain reaction results and microarray profiles of 13 genes examined across a subset of 20 pairs of ovarian samples. These findings will help to improve applications of microarrays in future ecotoxicological studies.
authors.affiliation: Ecological Exposure Research Division, National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, Ohio 45268, USA. wang.rong-lin@epa.gov
authors.forename: Rong-Lin
authors.initials: RL
authors.lastname: Wang
authors.forename: Adam
authors.initials: A
authors.lastname: Biales
authors.forename: David
authors.initials: D
authors.lastname: Bencic
authors.forename: David
authors.initials: D
authors.lastname: Lattier
authors.forename: Mitch
authors.initials: M
authors.lastname: Kostich
authors.forename: Dan
authors.initials: D
authors.lastname: Villeneuve
authors.forename: Gerald T
authors.initials: GT
authors.lastname: Ankley
authors.forename: Jim
authors.initials: J
authors.lastname: Lazorchak
authors.forename: Greg
authors.initials: G
authors.lastname: Toth
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D004784:Environmental Monitoring / Q000379:methods*; D004997:Ethinyl Estradiol / Q000633:toxicity; D017316:Fadrozole / Q000633:toxicity; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D010053:Ovary / Q000187:drug effects / Q000378:metabolism; D013737:Testis / Q000187:drug effects / Q000378:metabolism; D014158:Transcription, Genetic; D014204:Trenbolone Acetate / Q000633:toxicity; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D014874:Water Pollutants, Chemical; D029961:Zebrafish Proteins; D004997:Ethinyl Estradiol; D017316:Fadrozole; D014204:Trenbolone Acetate