treatment: Control (PBS) time: 18wks tissue: whole brain
Biomaterial provider
NOAA Fisheries, Northwest Fisheries Science Center
Growth protocol
Wild-type zebrafish (Danio rerio, AB strain) were obtained from Oregon State University, Sinnhuber Aquatic Research Laboratory (Corvallis, OR) and maintained at the Northwest Fisheries Science Center (NWFSC) in a ZebTec stand alone recirculating zebrafish rack system (Techniplast, Exton, PA). Fish were maintained at 26_C, kept on a 12h-12h light-dark cycle, and fed daily with commercially available fish feed.
Extracted molecule
total RNA
Extraction protocol
All fish were euthanized by decapitation followed by rapid removal of the brain for RNA extraction. During brain removal surgeries, the skullcap was opened, and the brain removed, rinsed in ice cold PBS, weighed, and immediately frozen in liquid nitrogen. The length of brain removal surgeries, from euthanasia to freezing in liquid nitrogen, averaged 1.6 ± 0.5 min (mean ± SD, n = 43). The entire brain was removed after cutting the brain stem and at the optic nerves as close to the eyes as possible. Total RNA was isolated from zebrafish brains using the miRNeasy Mini Kit (Qiagen Inc., Valencia, CA) according to the vendor’s defined method, and stored at -70_C. The quantity (ng/uL) of RNA was determined measuring the OD260 with a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and the RNA purity was assessed measuring OD260/280 and OD260/230 ratios. RNA integrity (quality) was characterized using the Agilent RNA 6000 Nano Kit with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only total RNA samples with appropriate size distribution, quantity, and A260/A280 as well as A260/A230 ratios of 1.8 – 2.1 were used for microarray-based analysis.
Label
Cy3 fluorescent label
Label protocol
For microarray hybridization, RNA was processed for hybridization to Agilent Zebrafish Gene Expression Microarray (V2) through a core facility in the Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health at the University of Washington according to the manufacturer's instructions.
Hybridization protocol
The RNA samples were labeled and prepared for hybridization onto a Zebrafish (V3) Gene Expression Microarray (Agilent Technologies, Inc. Santa Clara, CA) using the manufacturer’s established protocols. Hybridization and washing of these arrays was accomplished using HS 400 Pro hybridization and wash stations (Tecan Sysems, Inc., San Jose, CA).
Scan protocol
Arrays were scanned using an Agilent DNA Microarray Scanner (Agilent Technologies, Inc. Santa Clara, CA) through the University of Washington Functional Genomics Laboratory in the Center for Ecogenetics and Environmental Health (http://depts.washington.edu/ceeh/ServiceCores/FC1/FC1.html).
