Dichlorvos exposures were conducted for 24 h using control (no toxicant) plus the high, mid, and low concentrations of dichlorvos in the flow-through aquaria with concentrations maintained for the duration of the exposure. Fasting exposures were conducted using a control set with normal feeding (two times per day), a 24 h fasted group and a 50 h fasted group in the same flow-through aquaria as used previously. No toxicants were introduced during the fasting portion of this study.
Growth protocol
All exposures were conducted in 5-gallon glass aquaria adapted for flow-through use (60 mL/min; 5.4 turnovers/day) and maintained at 25°C with a 12h:12h (light:dark) photoperiod.
Extracted molecule
total RNA
Extraction protocol
Fish livers were homogenized in Trizol then total RNA was isolated from the homogenate following the Trizol manufacturer protocol (Invitrogen, Carlsbad, CA) followed by column purification with RNeasy Mini kits (Qiagen, GmbH, Germany) to remove salt and organic solvents. Total RNA quality and quantity were evaluated using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA) and verified using the NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 1.0 ug pooled RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were verified with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 uL containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 uL of 2x Agilent GEx hybridization buffer HI-RPM was added to the fragmentation mixture to stop the reaction. For each reaction 100 uL was hybridized to an Agilent Zebrafish custom Genome Oligo Microarrays (G2517A001, design 017662) for 17 hours at 65°C in a rotating Agilent hybridization oven (10 rpm). 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), 10 seconds with acetonitrile, and 30 seconds with Stabilization and Drying solution (Agilent) then scanned immediately.
Scan protocol
Dichlorvos exposure slides were scanned twice immediately after washing using a GenePix 4200 AL with a scan resolution of 5 um, standard green filter, PMT gain set at 400 and scan power set to 30% (first) then 100% (second). NOTE: GenePix scans are combined such that any probes which are offscale in the 100% scan are replaced by probes from the 30% scan. Fasting slides were scanned immediately after washing using an Agilent C2505C scanner following protocol GE1-107-Sep09 with default settings.
Data processing
Dichlorvos exposure study array scanned images were analyzed with GenePix Pro 6.1.0.4 using GalFile Zv2_019161_D_20080131.gal. Instrument default setting settings were used with the following exceptions: Background Subtraction: width of background = 2 feature diameters; Resize Features: minimum diameter = 80%, maximum diameter = 110%; Feature Movement: maximum translation = 20 um; CPI threshold = 0 with local background subtraction. Fasting study array scanned images were analyzed with Agilent Feature Extraction Software using grid template 019161_D_2008131 with default settings.
