tissue: Liver background strain: C57Bl/6J gender: male genotype: WT+/+ treament: DEN treatment duration (months): 6
Treatment protocol
Diethylnitrosamine (DEN, C4H10N2O,) was intraperitoneally administrated to wild type and bid-deficient mice at the age of day 15 as previously described (Bai et al, American Journal of Pathology, 166: 1523-1532, 2005). Wild type mice were in C57Bl/6J background whereas bid-deficient mice had been backcrossed to C57Bl/6J background for at least 12 generations. Male mice were used because they were susceptible to this model of carcinogenesis. Untreated control mice in the same genetic background were age and sex-matched. All of the mice were bred in house. Mice were euthanized by cervical dislocation following anesthesia at an early stage (4-6 months) and at a later stage (10-12 months) following DEN treatment. Livers were examined and processed. The treatment of mice were approved by the IACUC of the University of Pittsburgh.
Extracted molecule
total RNA
Extraction protocol
Mouse livers were dissected and RNA was prepared. In brief, 20 to 50 mg of liver tissues were disrupted in Buffer RLT (provided in Qiagen RNEasy kit), and homogenized. One volume of ethanol (70%) was then added to the lysate, creating conditions that promote selective binding of RNA to the RNeasy membrane. The sample was then applied to the RNeasy Mini spin column. Contaminants were washed away using RW1 wash buffer (provided in the RNEasy kit). RNA was eluted in RNase-free water. All binding, wash, and elution steps were performed by centrifugation in a micro-centrifuge.
Label
biotin
Label protocol
Biotinylated cRNA was prepared using standard protocol.
Hybridization protocol
For hybridization, 15 to 20 µg of cRNA were fragmented by incubating in a buffer containing 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc at 95°C for 35 minutes. The fragmented cRNA were then hybridized with a pre-equilibrated Affymetrix mouse chip (U74Av2) at 45°C for 14-16 hours. After the hybridization cocktails were removed, the chips were then washed in a fluidic station with low-stringency buffer (6x SSPE, 0.01% Tween 20, 0.005% antifoam) for 10 cycles (2 mixes/cycle) and stringent buffer (100 mM MES, 0.1 M NaCl and 0.01% Tween 20) for 4 cycles (15 mixes/cycle), and stained with SAPE (Strepto-avidin Phycoerythrin). This was followed by incubation with biotinylated mouse anti-avidin antibody, and restained with SAPE.
Scan protocol
The chips were scanned in a HP ChipScanner (Affymetrix Inc, Santa Clara, CA) to detect hybridization signals.
Description
The RNA was extracted from two equally treated samples
Data processing
The CEL files generated from the microarray scanning were used as the raw data. Data quality was examined using the RMAExpress program (v.1.0.5) (http://rmaexpress.bmbolstad.com/). The density distribution along the Log2 scale was comparable among the samples, which indicated that the quality of the assay was acceptable for comparison. Microarray data from individual mouse livers within each treatment group were then averaged and the averaged values were used for the subsequently comparisons and pathway analysis. Significance of differential expression was assessed using un-paired t-test. The threshold for the significant changes was a difference larger than 25% for upregulated genes and larger than 20% for downregulated genes (fold of change >1.25 or <0.8, p<0.05). Pathway analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatics Resources 6.7 (https://david.ncifcrf.gov/), and Gene Set Enrichment Analysis (GSEA 2.1.)(http://software.broadinstitute.org/gsea/index.jsp), for the differentially expressed gene set and the total gene set, respectively. The pathway database is from the KEGG annotation (http://www.genome.jp/kegg/pathway.html).
