normalization data transformation protocol
Raw intensity values on the microarrays were normalized, modeled, and compared using default options within the dChip (DNA Chip Analyzer) software package. The invariant set normalization method was used to compare arrays to a selected baseline array (GeneChip with median intensity) and the PM-MM model-based expression index (MBEI) analysis was used for computing expression values for gene comparison. ID_REF = VALUE = dChip's PM-MM Model-Based Expression Index (MBEI)
array scanning protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G System.
Following fragmentation, 11 ug of cRNA were hybridized with rotation at 60 rpm for 16 hr at 45C on GeneChip Human Genome U133 Plus 2.0 Array using Affymetrix's FS450_0001 fluidics protocol.
Biotinylated cRNA from iHeps were prepared according to the standard Affymetrix protocol using the GeneChip 3' IVT Plus Reagent Kit (Expression Analysis Technical Manual, 2001, Affymetrix). Primary Hepatocyte double-stranded cDNA was synthesized using a T7- (dt)24 primer (Oligo) and reverse transcription (Invitrogen). Biotin-labeled cRNA was synthesized using the BioArray HighYield RNA Transcript Labeling Kit (Enzo Life Sciences).
nucleic acid extraction protocol
iHep RNA was prepared using the RNeasy Mini Kit (Qiagen) and treated with Optizyme Recombinant DNase I to remove genomic DNA (Fisher Bioreagents). Primary Hepatocyte RNA was extracted via TRIzol (Invitrogen) and purified using the RNeasy Kit (Qiagen) as per manufacturer’s instructions.