age: adult (6 - 9 months) chemical group: Ni dose: mid chemical concentration (measured): 51.0 mg/L tissue: whole organism
Treatment protocol
Exposures were conducted for 24 h using control (no toxicant) plus the high, mid, and low concentrations of each metal in the flow-through aquaria with metal concentrations maintained for the duration of the exposure.
Growth protocol
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
Whole frozen fish were pulverized under liquid nitrogen using a SPEX 6750 freezer mill (SPEX Sample Prep, Metuchen, NJ). Total RNA was isolated from the pulverized material using Trizol (Invitrogen, Carlsbad, CA) with an extra clarification centrifugation step to remove bone, scales, lipid and other debris, followed by column purification with RNeasy Mini kits (Qiagen, GmbH, Germany) to remove residual 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
Slides were scanned 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 45%.
Description
564-005-03-P3
Data processing
Scanned images were analyzed with GenePix Pro 6.1.0.4 using GalFile 017662_D_20070905_update8. 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.
