strain: Sprague-Dawley age: 6-week old gender: male tissue: liver carcinogen: test compound
Treatment protocol
Each experimental compound was administered to three SD rats. In the repeated administration groups, the compounds were administered three times after three hours and again after 24 hours. The rats were autopsied after 24 hours, and RNA was isolated from liver tissue
Growth protocol
The animals were housed in polycarbonate cages, with hardwood chips, in a room with 12/12 h light/dark cycles at a controlled temperature. They were given food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of NIFDS.
Extracted molecule
total RNA
Extraction protocol
Liver specimens were processed for RNA extraction. RNA was isolated using TRIzol (Invitrogen, MO), and quality control was performed using RNA 6000 Nano chips on an Agilent 2001 Bioanalyzer. Total RNA was extracted from frozen tissue using TRIzol (Invitrogen, Carlsbad, CA) and was purified with an RNeasy mini kit (QIAGEN, Hilden, Germany). Total RNA (1 µg) was amplified using the Affymetrix one-cycle cDNA synthesis protocol. (Agilent Technologies, Germany). Total RNA was extracted from frozen tissue using TRIzol (Invitrogen, Carlsbad, CA) and was purified with an RNeasy mini kit (QIAGEN, Hilden, Germany).
Label
biotin
Label protocol
Biotin-labeling following standard Affymetrix protocols.
Hybridization protocol
For each array, 15 µg of amplified biotin-cDNA was fragmented and hybridized to the Affymetrix Rat Genome 2302.0 GeneChip array (Affymetrix, Santa Clara, CA) for 16 h at 45°C in a rotating hybridization oven. Slides were stained with streptavidin/phycoerythrin and washed for antibody amplification.
Scan protocol
Arrays with hybridized targets were scanned using an Affymetrix GeneChip Analysis System (GC450 Fluidics Stations, a Hybridization Oven 640, a GC3000 7G scanner), and the scanned images were analyzed using GeneChip® Operating Software v1.4 (GCOS) (Affymetrix). Spots of poor quality, as determined by visual inspection, were excluded from further analysis.
Description
ME(3x300)_2
Data processing
Forty-one data were obtained for the training compounds in this study. We treated rats with one of three compounds or a control for each GTX and NGTX experimental group and measured 2 time points for each group. All experimental procedures were repeated three times. We excluded 1 file that was found to be a failure. Each ‘.CEL’ file obtained as a raw data file was standardized using the RMA (Robust Multi-array Average) algorithm. Filtering was based on the expressed values of the raw data; probes with one or more expression values in the percentile range of 20-100% (27,458 of 31,099 total chip probes) were selected.
