44 human liver tissue surgical samples (normal n=13; steatosis n=19; steatohepatitis n=12, 8/2 cirrhotic/non-cirrhotic) obtained from patients undergoing liver surgery for HCC, other malignancy/metastatic disease, benign tumors of the liver and organ dedicated to transplantation. Virtually normal control samples in this cohort were taken from patients undergoing surgical resection of liver metastasis, but the surrounding liver tissue used for the molecular analyses was normal, non-tumorous liver tissue without detectable pathological changes. The samples were provided by the Biobank at the Medical University of Graz.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the samples using TRI Reagent® (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer´s protocols. The RNA quality was analyzed using microcapillary electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, Böblingen, Germany). Only samples with RIN (RNA integrity number) of 5.0 or higher were subjected to gene expression array analysis.
Label
biotin
Label protocol
Total RNA was prepared for hybridization on Illumina human-6 v3 BeadChip arrays (Illumina) according to manufacturer’s recommendations. In brief, 300 ng/µl total RNA was used for cDNA synthesis, followed by the generation of biotin-labeled cRNA according to the MessageAmp II aRNA Amplification kit (Ambion).
Hybridization protocol
Hybridization is performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes. After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays are dried and scanned.
Scan protocol
Microarray scanning was done using a Beadstation array scanner, setting adjusted to a scaling factor of 1 and PMT settings at 430.
Data processing
Data processing was done using open source statistical tool lumi package from Bioconductor. Data extraction was done for all beads individually, and outliers are removed when > 2.5 MAD (median absolute deviation). All remaining data points are used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated. Finally data was transformed using quantile normalization.
