tissue: ovary age (month): 5 treatment: in water control treatment duration: 48 h experiment: TRB_Phase3 sampling date: SampYM2007_6 scan date: ScanYM2008_6 rna date: rnaYM2007_12 rna person: rnaB_ROBOT chip id: 251959710043_1_4
Treatment protocol
To initiate a typical experiment, four males and four females were placed together in a glass test tank holding 10 L of Lake Superior water (4 µM filtered, UV sterilized) or test chemical dissolved in Lake Superior water, which was continuously renewed at a rate of about 45 mL/min. There were two such tanks for each treatment condition. The animals were held at 25 +/- 0.5 °C under a 16:8 L:D photoperiod, and were fed brine shrimp to satiation twice per day. chemical abbreviations: BPA, bisphenol-A; EE2, 17a-ethynyl estradiol; FAD, fadrozole; FLU, flutamide; GEM, Gemfibrozil; KTC, ketoconazole; PRO,prochloraz; RDX, hexahydro-1,3,5-trinitro-1,3,5-triazine; TB, 17 -trenbolone; TNT, 2,4,6-trinitrotoluene; TRI,trilostane; VIN, vinclozolin; WLSSD, effluent from Western Lake Superior Sanitary District
Growth protocol
Sexually mature adult fathead minnows (5-6 month old) were obtained from an on-site aquatic culture facility at the US EPA Mid-Continent Ecology Division, Duluth, MN, USA. Fish were held under a 16:8 light:dark photoperiod fed frozen adult brine shrimp (San Francisco Bay Brand, Newark, CA, USA). Fish survival, water temperature (mean ± SD; 25.0±0.3), and dissolved oxygen (mean ± SD; 7.0±0.2) were monitored daily over the duration of the exposure and did not vary significantly between treatment groups.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from ovary tissue of both fathead minnow using RNeasy kits (Qiagen, Valencia, CA, USA). RNA quality was evaluated using an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE, USA) and RNA was quantified spectrophotometrically (Nanodrop). Total RNA samples were stored at -80oC until used for microarray analyses.
Label
Cy3
Label protocol
cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturer's kits and protocols (Quick Amp Labeling Kit; Agilent, Palo Alto, CA)
Hybridization protocol
The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies, Palo Alto, CA) was used for microarray hybridizations following the manufacturer’s protocol and recommendations.
Scan protocol
An Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc.) was used to scan microarray images at 5 µm resolution
Data processing
Microarray image processing was performed using the latest Agilent's Feature Extraction software available at the time following the manufacturer’s protocol and recommendations. Data processing was conducted using R package Limma.
