Expression data from Wilson disease patients liver

The goal of the study was to analyze gene expression profle from the WD patients who underwent liver transplantation for acute or chronic liver failure. Control specimens were obtained from patients who underwent liver resections for other clinical reasons. We used microarrays (Affymetrix) to determine the gene expression profile in WD Liver sections were snap frozen in liquid nitrogen and stored. Total RNA was isolated using TRIZOL reagent (Invitrogen, Grand Island, NY) followed by RNeasy cleanup procedure (Qiagen, Valencia, CA). The integrity of isolated RNA was electrophoretically verified by ethidium bromide staining and by optical densities (OD) ratio (OD260nm/280nm>1.8). A total of 15 liver RNA samples (8 biological replicates for control liver and 7 for WD patients) were examined for RNA integrity and concentration on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6.000 LabChip Kit (Agilent Technologies) according to the manufacturers instructions. Affymetrix GeneChip analysis was conducted at the microarray core facility of the Interdisziplinäres Zentrum für klinische Forschung (IZKF), Leipzig (Faculty of Medicine, University of Leipzig). 2 μg of total RNA were used to prepare double-stranded cDNA (Superscript II, Life Technologies, Gaithersburg, MD) primed with oligo-dT containing an T7 RNA polymerase promoter site (Genset SA, Paris, France). cDNA was purified by phenol- chloroform extraction before in vitro transcription using the IVT labeling kit (Affymetrix, Santa Clara, CA, USA) to synthesize cRNA. After the in vitro transcription, unincorporated nucleotides were removed using the RNeasy kit (QIAGEN). The cRNA was fragmented and hybridized to two different (technical replicates) Human Genome U133 Plus 2.0 (Arrays Affymetrix). The washing and staining of the probe array was performed according to the manufacturer’s instructions. The array was scanned with a third generation Affymetrix GeneChipScanner 3000.