strain: C57Bl6 gender: male cell type: primary hepatocytes
The primairy mouse hepatocytes were exposed as to 10 μM cyclosporine A or 0.5 % DMSO as a vehicle control for 48 hours.
Cells from three independent biological replicates with viability > 80 were used and cultured on collagen gel-precoated six-well plates at a density of 6.5 105 cells/ml. Cells were allowed to attach for 2 to 4 h at 37°C in a humidified chamber with 95:5% air/CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 0.5 U/ml insulin, 7 ng/ml glucagon, and 2% penicillin/streptomycin (5000 U/ml penicillin; 5000 μm/ml streptomycin). After attachment, medium containing unattached cells and debris was removed by washing, and the cultures were overlaid with a second collagen layer to form a collagen-collagen sandwich culture. Primary cultures of mouse hepatocytes were cultured at 37°C in a humidified chamber with 95:5% air/CO2 in serum-free culture medium supplemented with 0.5 U/ml insulin, 7 ng/ml glucagon, 7.5 μg/ml hydrocortisone, and 2% penicillin/streptomycin (5000 U/ml penicillin; 5000 μm/ml streptomycin). Cells from three independent biological experiments (each from a different animal) were harvested at 48 h after isolation for gene expression analysis.
Total RNA was isolated after 48 hours of incubation with cyclosporin A or DMSO. Total RNA was isolated from cells using miRNeasy mini Kit (Qiagen Westburg bv, Leusden, the Netherlands) according to the manufacturer's instructions and followed by a DNAse I (Qiagen Inc.) treatment. RNA quantity was measured on a spectrophotometer and quality was determined on a BioAnalyzer (Agilent Technologies, Breda, the Netherlands). Only RNA samples which showed clear 18S and 28S peaks and with a RIN level higher than 8 were used.
cRNA targets were prepared according to the Affymetrix protocol.
The targets were hybridized according to the manufacturer's recommended procedures on high-density oligonucleotide GeneChips (Affymetrix Mouse Genome 430 2.0 GeneChip arrays).
The GeneChips were washed and stained using a fluidics station by Affymetrix and scanned in an Affymetrix GeneArray scanner.
SAMPLE 1 Gene expression data from Primary mouse hepatocytes exposed to 10µM Cyclosporin A or 0.5 % DMSO
First, the quality of all arrays was inspected using arrayQC, an in-house quality control pipeline that generates virtual images, boxplots, correlation plots, clustering images, MvA and PCA plots. Spike-ins were used for all arrays as an extra quality check. Within this dataset, no arrays were technically deviating. The CustomCDF annotation (Affymetrix mouse4302) (Version 11.0.1, ENTREZG) from BrainArray Microarray were used. The Affymetrix .CEL files were imported in R 2.11.0 using the affy library in BioConductor. All probes were reannotated using the BrainArray customCDF files (EntrezGene, version 11.0.1), followed by a RMA normalization step. Prior to the statistical analysis all probesets (excluding positive and negative controls spotted in the array) had to pass a detection filter criteria [Present/Marginal >= 2 out of 3 biological replicates per condition, control or treated with CsA], resulting in a total of 10,848 reporters.