Feeding study, exposure from gestation day 6 through to post natal day 92
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from PND92 testis of individual Wistar Han IGS rats of the mid dose (M500, P500, T500 ppm) and high dose (M2000, P2500, T1800 ppm) treatment groups using TRI Reagent (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer’s protocol and subjected to quality control measures before application to microarrays. For quality control, RNA A260:A280 ratios were assessed via NanoDrop Fluorospectrometer (NanoDrop Technologies, Inc. Wilmington, DE). RNA absorbance readings with a range 1.8 – 2.1 were followed with DNase treatment, Total RNA Cleanup (Qiagen RNeasy), and then checked for RNA quality using the model 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA). Those samples with a ratio of 28S:18S rRNA >1.6 were accepted for subsequent use in DNA microarrays. RNA was stored at -80ºC until labeling for microarray hybridization.
Label
biotin
Label protocol
Five micrograms or purified total RNA from each liver or testis of 3-7 individual rats per treatment group was hybridized to Affymetrix GeneChip® Rat Genome 230 2.0 plus microarrays according to the Affymetrix GeneChip Expression Analysis Technical Manual (www.affymetrix.com). Briefly, after purified RNA passed quantity and quality assessment, double stranded cDNA was synthesized from RNA using reverse transcriptase and an oligo-dT primer. Biotinylated RNA (labeled cRNA) served as the microarray target. cRNA was fragmented using heat and magnesium (Mg+2) reducing the cRNA to 25-200 base fragments to facilitate efficient and reproducible hybridization.
Hybridization protocol
cRNA was combined with the hybridization cocktail, containing 3nM B2 control oligo, 10 mg/ml Herring Sperm DNA, 50 mg/ml BSA, 100% DMSO, and 2X hybridization buffer (NaCL 5M, MES hydrate Sigma Ultra, MES Sodium Salt, EDTA Disodium Salt 0.5M, 10% Tween-20). GeneChips were hybridized at 45ºC for 16 h. After hybridization the chip was washed and stained with fluorescent streptavidin-phycoerythrin, binding to biotin for detection.
Scan protocol
Signal amplification using anti-Streptavidin antibody (goat) and biotinylated goat IgG antibody was used to bind biotin and provide an amplified fluor that emitted light when the chip is scanned with the GeneChip Scanner 3000.
Description
P500_04
Data processing
To minimize non-biological factors, e.g. total amount of target hybridized to each array, signal values for each hybridization were multiplied by a scaling factor (SF) to get a trimmed mean intensity equal to 500. Converted .cel files were loaded into JMP Genomics (SAS Inc., Cary, NC), Log2 transformed, normalized using interquartile normalization, and analyzed for significant changes in transcript levels through row-by-row modeling using one-way ANOVA. For initial exploratory analysis, principle component analysis (PCA) was applied using JMP Genomics. Comparisons were made between controls and each treatment group with statistical cut-offs applied at a p-value adjusted false discovery rate (FDR) of 25% for testis (p<0.000229), and an absolute difference of |1.2| or greater. Probe sets representing transcribed loci, unknown genes, and image clones were removed from the final list of each analysis.
