Per concentration and replicate, 24 newly fertilized embryos (<2hpf) were exposed to EC10 and EC20 concentrations (for details, see design) of EE2, BPA, methylparaben, propanil, flutamide, linuron and prochloraz for 48 hours in 96 well plates. After exposure, each group of 24 fish embryos was pooled together, snap-frozen over dry ice and stored at –80°C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from frozen, pooled embryos by homogenization in TRIreagent (Sigma-Aldrich, T9424) followed by extraction with chloroform and ethanol and final purification using RNeasy Mini Spin Columns (QIAGEN, Hilden, Germany) according to the manufacturers’ protocols. RNA concentration was measured using a NanoDrop 1000 (Thermo Fisher Scientific, Schwerte, Germany) and RNA quality was verified on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA).
Label
Cy3
Label protocol
We carried out cDNA synthesis, cRNA Cy3 labeling, amplification and hybridization according to the manufacturer’s protocols (Quick Amp Labeling Kit; Agilent Technologies), using 500ng RNA as starting material.
Hybridization protocol
Hybridization was carried out according to the Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies)
Scan protocol
Microarrays were scanned immediately after wahsing with the Agilent Microarray Scanner (Agilent Technologies)
Data processing
Data were extracted and processed using Agilent Feature Extraction Software applying default parameters (protocol GE1-v5_95_Feb07, QC metric set GE1_QCMT_Feb07)
