Sprague Dawley rats Gender: male Weight: 250-350 g
Treatment protocol
The soman exposure paradigm utilized in this study was based on the model developed by Shih et al. (1991) and McDonough et al. (1998). Animals were pretreated with the oxime HI-6 (125 mg/kg, ip, obtained from the Walter Reed Army Institute of Research, Rockville, MD) 30 min prior to challenge with soman (180 μg/kg, sc; diluted with 0.9% sodium chloride; soman was obtained from the Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD). One minute after soman challenge, animals were treated with atropine methyl nitrate (2.0 mg/kg, im, Sigma-Aldrich, St. Louis, MO). Vehicle control animals (n=4; 2 euthanized at 1h, 1 euthanized at 12h, and 1 euthanized at 24h) received an equivalent volume of vehicle (0.9% sodium chloride), HI-6 and atropine. Naïve animals (n=3) were also included in the study and received no treatments.
Extracted molecule
total RNA
Extraction protocol
Frozen tissue was homogenized in Tri Reagent (Sigma Chemical Company, St. Louis, MO) and the RNA extracted according to the manufacturer's protocol. RNA was further purified using RNeasy columns (Qiagen, Valencia, CA). The quality and amount of RNA was monitored throughout processing with an Agilent Bioanalyzer (Agilent, Palo Alto, CA) and a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Nanodrop Technologies, Rockland, DE).
Label
biotin
Label protocol
10 µg of mRNA was used to generate first-strand cDNA using a T7-linked oligo(dT) primer. After second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Kits from Affymetrix).
Hybridization protocol
Spike controls were added to 15 µg fragmented cRNA before hybridization (36h) using 10 µg of cRNA. Arrays were washed and stained with streptavidin-phycoerythrin before being scanned on an Affymetrix GeneChip Scanner.
Scan protocol
After scanning, array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches on the chip surface. The 3'/5' ratios for GAPDH and B-actin were checked for quality. BioB, BioC, BioD and CreX spike controls were also examined to determine sample quality. All arrays were scaled to a target intensity of 150 using Affymetrix GeneChip Operating System (GCOS) software.
Description
Individually housed; controlled temperature and humidity;12h light/12h dark cycle; food and water ad libitum.
Data processing
Raw signal intensities were imported into Partek Genomics Solutions v6.3 (Partek, St. Louis, MO) and normalized using the robust multi-array averaging (RMA) algorithm (Irizarry et al., 2003)
