Sodium arsenite at prescribed concentrations was added to food, which was repelleted, or sodium arsenite was added to water. Injections were intraperitoneal in a normal saline solution.
Growth protocol
6wk male C57BL/6 male mice were acclimated to their respective diets for 2 wk prior to treatments.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from frozen lung samples and homogenized using QiashredderTM and RNeasy® Mini Kits (Qiagen, Valencia, CA) per manufacturer's protocol. Contaminating genomic DNA was removed using DNA-free kits (Ambion, Austin, TX) and total RNA was quantified with a NanoDrop®ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE). RNA quality was determined with the RNA 6000 LabChip kit Nano Assay (Agilent Technologies, Inc., Santa Clara, CA).
Label
biotin
Label protocol
Total RNA was first reverse-transcribed using T7-oligo(dT) promoter primer in the first-strand cDNA synthesis reaction. Following RNase H-mediated second-strand cDNA synthesis, the double stranded cDNA was purified and served as a template in the subsequent in vitro transcription (IVT) reaction.
Hybridization protocol
The IVT reaction was carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling. Biotinylated cRNA (15 ug) targets were then cleaned up, fragmented, and hybridized to GeneChip® array during the overnight incubation at 45oC in rotating hybridization oven.
Scan protocol
After hybridization, the arrays were stained with streptavidin-phycoerythrin in the GeneChip Fluidics station and then scanned using Affymetrix GeneChip Scanner (laser filter set at 570 nm; pixel size 2.5 um). The array image data were acquired, and the fluorescent signal intensities were quantified using Affymetrix® GCOS v. 1.2 software with following settings of quantitation parameters: Alpha1=0.05, Alpha2=0.065, Tau=0.015, Gamma1H=0.0045, Gamma1L=0.0045, Gamma2H=0.006, Gamma2L=0.006, Perturbation=1.1, Target Intensity=150.
CEL file data were normalized using RMA as implented in Bioconductor simpleaffy. Genes were selected by Rank Product (Breitling, R., 2004, FEBS Letter, 57383-92) implemented in Bioconductor RankProd at p < .05 and by ANOVA.
