Dosing capsules were filled by weighing pure RDX into size 9 gel capsules (Torpac, Fairfield, NJ). Capsules were then individually stored in dark glass sample bottles for up to two days in a refrigerator until required for dosing. At the time of dosing animals weighing 300-400 g were administered a one time dose of pure RDX in a gel capsule according to the manufacturer's instructions. Doses were calculated, based on previous work, so as not to induce seizure, and animals were observed over the first hour to confirm lack of seizure.
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 overnight (16hr) hybridization 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
pair-housed; one week acclimatization period; 12/12 light/dark cycle; food and water provided ad libitum.
Data processing
Data was imported into Gensifter (VizX Laboratories, Seattle, WA) (www.gensifter.net) and normalized using Robust Multiarray Averaging (RMA). Data was also imported into Partek Genomics Solutions (Partet, St. Louis, MO), normalized using RMA, and visualized using principal component analysis (PCA).
