ID_REF = VALUE = Log2 GCRMA Signal
Affymetrix 7G Scanner/GCOS
Partek Genomics Suite GCRMA Algorithm
Protocol follows the Affymetrix Hybridization, Wash and Stain kit for Eukaryotic Target Hybridization for 49 Format Array using 15 ug fragmented cRNA and 45C hyb temp for 16 hours at 60 rpm. Washed with Affymetrix 450 Fluidics Station using FS450_001 script.
Larvae were centrifuged for 10 min. at 13,000 rpm to separate larvae from exposure water, and pellets containing larvae were stored at <-80C until RNA extraction was performed. Total Larval RNA was extracted using the Rneasy mini extraction kit for animal tissues (Qiagen, Valencia, CA) and quantified using a spectrophotometer (Nanodrop, Wilmington, DE).
Larval zebrafish (72 hpf) were exposed for 96 h in 200ml fish water containing appropirate amount of SSRI stock (i.e. fluoxetine or sertraline). There were four SSRIs treatments (25 and 250 ug/L fluoxetine and 25 and 250 ug/L sertraline) and one control (no SSRIs) with triplicate beakers and each beaker contained about 100 larval fish. During exposure for 96 hours, beakers were kept covered to prevent water evaporation and fish were not fed (i.e., fish consumed their yolk sac).
Affymetrix 3' labeling kit 5 ug total RNA
Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained at the Center for Environmental Biotechnology at the University of Tennessee. Fish husbandry, spawning, and experimental procedures were conducted with approval from the UT Insititutional Animal Care and Use Committee (Protocol #1690-1007). Water for holding fish and conducting experiments (hereafter referred to as fish water) consisted of MilliQ water (Millipore, Bedford, MA) with ions added: 19 mg/L NaHCO3, 1 mg/L sea salt (Instant Ocean Synthetic Sea Salt, Mentor, OH), 10 mg/L CaSO4, 10 mg/L MgSO4, 2 mg/L KCl. Embroyos were obtained by spawning adult fish with no history of contaminant exposure. Fertilization of embryos took place at the same time (<15 minutes), such that larvae used in experiments were of similar age at the time of exposure. All activities (maintenance of adult fish, spawning, and experiments) were conducted in an environmental chamber with a temperature of 27 +/- 1 C and 14:10h light:dark photoperiod.