Zebrafish embryos were exposed to 0, 0.3, 3, or 30 ppb atrazine from approximately 1 hpf through 72 hpf. 50 embryos were pooled per sample (considered as one biological replicate). Three biological replicates completed (n=3). Larvae were homogenized in Trizol (Life Technolgies, Carlsbad, CA) and flash frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Growth protocol
Zebrafish (wild-type AB strain) were housed in a Z-Mod System (Aquatic Habitats, Apopka, FL) on a 14:10 hour light:dark cycle and maintained at 28ºC ± 1°C with a pH of 7.0-7.2 and conductivity range of 470-520 µS.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using an RNeasy Mini Kit (Qiagen Sciences, Maryland, USA) following established protocols. RNA concentrations were measured by using a NanoDrop 1000 spectrophotometer and the quality was verified by absorbance ratio 260/280 nm. Total RNA was converted to cDNA following established laboratory protocols.
Label
Cy3
Label protocol
cDNA was labeled using the NimbleGen One-Color DNA Labeling Kit, following the manufacturer's instructions (Roche NimbleGen, Madison, WI). Quality (Yield and specificity) of Cy3 labeled cRNA was measured on Drop® ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE).
Hybridization protocol
Cy3-labeled cDNA samples were mixed with hybridization mix containing 2X Hybridization Buffer, Hybridization Component A, and Alignment Oligo using NimbleGen Hybridization Kit, according to the manufacturers recommendations (Roche NimbleGen, Madison, WI). The samples were incubated at 95°C on a heat block for 5 miniutes for denaturation, and then placed on arrays and hybridized at 42°C overnight on a Thermobrite StatSpin® (Abbott Molecular, Inc., Abbott Park, IL) . Following the hybridization, array slides were washed using Wash Buffer I, II, and III in NimbleGen Wash Buffer Kit (Roche NimbleGen, Madison, WI), and then air-dried briefly.
Scan protocol
Array slides were scanned on a GenePix 4000B microarray scanner with one-color scan setting at 5 microns.
Description
Gene expression 72 hpf following embryonic exposure (1-72 hpf)
Data processing
Array images were extracted with NimbleScan software (Roche NimbleGen, Madison, WI) in which fluorescence signal intensity values were normalized using quantile normalization and gene calls were generated using the Robust Multichip Average (RMA) algorithm following manufacturer recommendations.
Transcriptome Alterations Following Developmental Atrazine Exposure in Zebrafish Are Associated with Disruption of Neuroendocrine and Reproductive System Function, Cell Cycle, and Carcinogenesis
