Identification and characterization of androgen responsive biomarker genes in zebrafish embryos

Exposure experiments with the non-aromatizable fish androgen, 11-ketotestosterone in the range of 0.05-5000 nM were conducted in order to identify potential androgen-responsive genes in zebrafish embryos by microarray analysis. Zebrafish embryos were treated from 96 hpf to 120 hpf in a single experiment with three controls (embryonic medium). Microarray studies were performed using a Custom 8x60k Gene Expression Microarrays (Amadid G4102A, based on the ensembl zebrafish genome version 3) from total RNA with low input labelling and hybridization kit (Agilent Technologies) and one-color design according to the manufacturer instructions. Fluorescent intensities of individual microarray spots were extracted using the Agilent Feature Extraction software. Raw microarray data were converted to log2 values and quantile-normalized. For further statistical analysis, fold changes in relation to the mean of the controls were calculated for each treatment. Zebrafish embryos were treated from 96 hpf to 120 hpf in a single experiment with three controls and 11 concentrations of 11-ketotestosterone ranging from 0.05-5000 nM.