normalization data transformation protocol
Affymetrix CEL files were each checked on quality, including RNA degradation control, correlation and clustering. All quality checks were within acceptable limits, according to Affymetrix standards. After quality control the files were normalized with the Multichip Average (RMA) procedure (Irizarry et al., 2003), using the custom chip description files (CDFs) as described by de Leeuw et al. (2008). Of the hybrid probe-set definitions included in the custom annotation, only the 16,331 probe sets selected according to Dai et al. (2005) and the 4,648 Affymetrix probe sets corresponding to an Entrez Gene ID were used for further analysis, giving a total of 20,979 probe sets. Principal Component Analysis (PCA) was used to rule out effects of the experimental variables. For detecting significantly regulated genes, the microarray analysis of variance (MAANOVA) package in R was used (R version 2.9.2, www.r-project.org). For the analyses on significantly regulated genes per compound between treated and control samples, an F1-test was used. Gene-specific P-values were corrected with a Benjamini- Hochberg false discovery rate (FDR) with a cut-off at 0.05 (Benjamini and Hochberg, 1995). ID_REF = VALUE = log2 RMA signal
array scanning protocol
After hybridization the array chips were washed and stained with a Genechip Fluidics Station 450 and scanned using the Affymetrix gene chip scanner 3000.
hybridized to the Affymetrix Mouse Genome 430 2.0 GeneChip arrays, according to the manufacturer’s instructions.
Labeled RNA was prepared using the Affymetrix gene chip 3’IVT express kit
sample treatment protocol
Forty-six hours after isolation, hepatocytes were exposed to either 300 µM DCF or the solvent DMSO.
Primary mouse hepatocytes were isolated from 8-10 weeks old male C57BL/6 mice by a modified two-step collagenase perfusion technique (collagenase type IV, Sigma-Aldrich, Zwijndrecht, The Netherlands), as described by van Kesteren et al. (2011). In short, hepatocytes suspensions with at least 80% viability were seeded onto plates coated with collagen gel and after attachment overlayed with a second layer of collagen gel, to form a sandwich configuration. Cells were kept in serum-free medium and the culture medium was changed daily until exposures were performed.
nucleic acid extraction protocol
RNA was extracted using TRIzol (Invitrogen) and purified using a RNeasy mini kit (Qiagen), both according to the manufacturer’s protocol.