Fluidics station 400

Total RNA was extracted from 7 days vehicle or of TGF-β1 treated cells using RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Biotinylated cRNA were prepared according to the standard Affymetrix protocol from total RNA (Expression Analysis Technical Manual, 2001, Affymetrix, Santa Clara, CA USA). Following fragmentation, cRNA was hybridized on GeneChip Rat Genome 230 2.0 Array (experiment 1) as well as on GeneChip Rat Expression 230A Array (experiment 2). For both experiments, GeneChips were washed and stained in the Affymetrix Fluidics Station 400 and then scanned using the Hewlett-Packard GeneArray Scanner G2500A. The expression profiling and data analysis of the two independent experiments were carried out at the competence centre for fluorescent bioanalytics (KFB; Regensburg, Germany).

An aliquot of 5 μg of high-quality total RNA from each sample was used to synthesize cDNA and biotinylated cRNA utilizing the Affymetrix GeneChip® Expression 3’Amplification One-Cycle Target Labeling and Control Reagent kit according to manufacturer’s instructions. Biotinylated cRNA was hybridized to Affymetrix Gene-Chips HG U133 Plus 2 (Affymetrix, Santa Clara, CA), washed, stained on the Affymetrix Fluidics station 400 and scanned with a Hewlett Packard G2500A Gene Array Scanner following Affymetrix instructions. All arrays used in the study passed the quality control set by Tumor Analysis Best Practices Working Group [24 ].