|Application:||Gene expression microarray analysis|
|Number of samples:||24|
|Release date:||Nov 22 2017|
|Last update date:||Feb 21 2018|
|Dataset link||Microarray-based transcriptomic responses of zebrafish embryos exposed to 2 µM TDCIPP from 0.75 hpf to 2 and 6 hpf|
Zebrafish embryos were exposed to 0.1% DMSO or 2 µM TDCIPP from 0.75 hpf. Embryos (25 per replicate) were collected at either 2 or 6 hpf, transferred from beakers to 2-mL cryovials, snap-frozen in liquid nitrogen, and stored at -80ºC. These experiments resulted in three independent replicate samples for each time point and treatment group, resulting a total of 12 samples for RNA extractions. Total RNA was extracted from pooled embryos using a SV Total RNA Isolation System (Promega). After elution of RNA in 100 μL nuclease-free water, total RNA concentrations, 260/280 ratios, and 260/230 ratios were quantified using a NanoDrop ND-2000 spectrophotometer (ThermoFisher Scientific) and then stored at -80°C; total RNA quality was also confirmed using an Agilent 2100 Bioanalyzer system. Total RNA samples were amplified and biotinylated using GeneChip WT PLUS Reagent Kit (Affymetrix). Briefly, 100 ng of total RNA per sample was reverse-transcribed into ds-cDNA using NNN random primers containing a T7 RNA polymerase promoter sequence; cDNA quality was then confirmed using an Agilent 2100 Bioanalyzer system. T7 RNA polymerase was then added to cDNA samples to amplify RNA, and then RNA was reverse-transcribed to ss-DNA and degraded using RNase H. ss-DNA molecules were then fragmented and terminally labelled with biotin. Amplified and labeled samples were hybridized onto duplicate Zebrafish Gene 1.0 ST Arrays (Affymetrix) for 16 h at 45°C using a GeneChip Hybridization Oven 640 and a GeneChip Hybridization, Wash, and Stain Kit (Affymetrix); these arrays were constructed by Affymetrix based on the danRer6/Zv9 genome build and contained 1,255,682 probes representing 59,302 unique genes. Hybridized arrays were washed and stained using GeneChip Fluidics Stations 450 (Affymetrix). Arrays (24 total) were then scanned using a GeneChip Scanner 3000 7G system and computer workstation equipped with GeneChip Command Console 4.0 software (Affymetrix).