Treatment chemicals were dissolved in dimethylsulfoxide (DMSO) and added to embryo media between 72 and 96 hpf
Growth protocol
Adult wild-type zebrafish (Danio rerio) from a commercial supplier (EkkWill, Gibsonton, FL) were maintained in 30 gallon aquaria at 28.5 degrees Celsius in a 14:10 hour light: dark cycle. Embryos were collected by natural spawning, washed and incubated, 100 to 120 embryos/plate in 150mL petri plates in 50mL egg water (Westerfield 2000) at 28.5 degrees Celsius in a 14:10 hour light: dark cycle.
Extracted molecule
total RNA
Extraction protocol
~30 larvae/sample were collected at 96 hpf in 1.5ml microfuge tubes, quick-frozen on dry ice, and stored at -70 degrees C for RNA extraction. Pooled frozen embryos were homogenized in Tri Reagent (Sigma), according to the manufacturer’s protocol to extract total RNA. RNA was resuspended in Diethylpyrocarbonate (DEPC) -treated water and treated with DNaseI (Roche, Indianapolis, IN). Quantity and quality were confirmed by UV spectrophotometry and gel electrophoresis. RNA was re-purified using the RNeasy kit according to manufacturer’s instructions (Qiagen, Valencia, CA)
Label
biotin
Label protocol
Total RNA (1 ug) was reverse transcribed using One Cycle cDNA Synthesis kit (Affymetrix, Santa Clara, CA) and the obtained double-stranded cDNA was purified with GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). The obtained cDNA was used as a template for in vitro transcription using GeneChip IVT Labeling Kit (Affymetrix, Santa Clara, CA). The obtained Biotin-labeled cRNA was purified using GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA)
Hybridization protocol
Purified cRNA was fragmented and hybridized (15 ug) to the GeneChip Zebrafish Genome Array (Affymetrix, Santa Clara, CA) for 16 hours in GeneChip Hybridization oven 640 at 45oC with rotation (60 rpm). The hybridized samples were washed and stained using Affymetrix fluidics station 450 with streptavidin-R-phycoerythrin (SAPE) and the signal was amplified using a biotinilated goat anti-streptavidin antibody followed by another SAPE staining (Hybridization, Washing and Sataining Kit, Affymetrix, Santa Clara, CA).
Scan protocol
Microarrays were immediately scanned using Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix, Santa Clara, CA).
Description
W252C
Data processing
Gene expression levels were generated using the MicroArray Suite 5.0 (MAS5) algorithm in Affymetrix Gene Expression Console, scaling to a global mean intensity of 500.
Submission date
Nov 06, 2012
Last update date
Jun 25, 2019
Contact name
Boston University Microarray and Sequencing Resource
