Zebrafish embryos at <2 hours post fertilization (hpf) were developmentally treated with 0, 0.3, 3, or 30 ppb atrazine until 72 hpf. Following the atrazine exposure, the embryos were washed and transferred to clean aquaria water with no atrazine treatment. Fish were grown until 5-6 months of age at which brains were subjected to RNA extraction and then cDNA synthesis for further microarray analysis.
Growth protocol
Zebrafish (AB stain) were cultured in a Z-Mod System (Aquatic Habitats, Apopka, FL) under a 14:10 light:dark cycle at 28℃. Water quality parameters (conductivity, pH, and temperature) were regulary checked.
Extracted molecule
total RNA
Extraction protocol
Zebrafish brains dissected at 5-6 months of age were homogenized in Trizol Reagent (Life Technologies, Carlsbad, CA) and stored at -80ºC until RNA isolation. Total RNA was extracted according to established protocols as descrived in Peterson and Freeman (2009) and then purified using the RNeay Mini Kit (Qiagen, Valencia, CA) following manufacturer’s recommendation. Quality and quantity of purified RNA samples were checked on the NanoDrop® ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE).
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 Nimblegen MS 200 microarray scanner with one-color scan setting (Scan region, 61 x 21.6 mm; Scan resolution, 2um; Tiff file dyamic range, 20 bit; Dye channel, Green; Green PMT gain, 100%).
Description
Gene expression in 5-6 months aged female brain after a developmental exposure to 30ppb atrazine
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.
