Four replicates were used per treatment with ten fish per replicate. Both fathead minnow and zebrafish investigation included control, 0.9, 1.8, 7.0 and 13.8 mg/L treatments.
Growth protocol
Control and test waters were Vicksburg, MS, USA tap water dechlorinated via activated charcoal filtration. Exposures were conducted in an environmental chamber at a temperature of 25 °C and light:dark cycle of 16h:8h. Each replicate was housed in individual beakers where dissolved oxygen concentrations were ensured via slight aeration (approximately one bubble per second) into each beaker with a single glass pipette connected to a constant air flow. Fish were exposed in 300 ml high-form lipless beakers. The exposures were static renewal, renewed once after 48-h. Fish were fed three hours before the 48-h water renewal. FHM were fed brine shrimp (Artemia sp.) nauplii, and ZF were fed ARTEMAC-0 fish food (20-80 µM diameter, Aquafauna Bio-Marine, Inc., Hawthorne, CA, USA).
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the RNeasy mini kit (QIAGEN, Valencia, CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
For ZF, 1650 ng of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom-designed Agilent test array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37°C GE Wash buffer 2 (Agilent), 30 seconds with Acetonitrile, and dried with Agilent Stabilization and Drying Solution.
Scan protocol
Slides were scanned immediately after washing on a GenePix 4200AL scanner at PMT gain level 420 using default setting (Scan resolution 5um, One channel-Green, Laser power 100%).
The scanned images were analyzed with GenePix Pro 6.1 (Molecular Devices, Sunnyvale, CA) using default parameters and an Agilient-provided GEML grid file to obtain signal intensities. GeneSpring GX (Agilent Technologies) was used to normalize data by 75th percentile shift and conduct baseline transformation to the median of all samples. Statistical analysis was also performed in GeneSpring utilizing data from all microarray probes and consisted of one-way ANOVA with Benjamini-Hochberg multiple-tests correction to test for significant differential expression of microarray targets.
Comparison of genomic, morphological, and behavioral responses among fathead minnow (Pimephales promelas ) and zebrafish (Danio rerio) to acute RDX exposure
