Feature extraction v11

Processed microarray data (signal values calculated by Agilent Feature Extraction V11.5.1.1, quantile-normalized and log2-transformed) were downloaded from the Gene Expression Omnibus database (accession no. GSE53787). Gene set enrichment analysis (Subramanian et al., 2005 (link)) was performed by using standard parameters (gene set permutation, signal-to-noise ratio as a ranking metric). Genes involved in regulation of TGFβ production (Gene ontology term GO:0032914) were downloaded from the QuickGO Browser (http://www.ebi.ac.uk/QuickGO/).

Before labeling, RNA concentration and integrity were measured using a Nanodrop™ spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and controlled on ‘Lab-on-Chip’ by a Bioanalyzer (Agilent Technologies, Stockport, UK), according to the manufacturer’s instructions. From each pool, 100 ng of RNA was converted into labeled cRNA with nucleotides coupled to a Cy3 fluorescent dye using the Low Input Quick Amp Labeling kit (Agilent Technologies, Palo Alto, CA, USA) following the manufacturer’s instructions. The quality and quantity of the resulting labeled cRNA were assessed using a Nanodrop™ spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). We have done two labelings/sample to obtain the quantity of Cy3 -labeled cRNA necessary for the hybridization (600ng).Each Cy3-labeled cRNA was hybridized to human microarray ship (Agilent Human V2 Microarrays, 8*60k, G4858A) for 17 h at 65°C, 10 rpm as described in the One-Color Microarray-Based Gene ExpressionAnalysis Protocol (Agilent). The hybridized arrays were then washed and scanned using an TECAN MS200 scanner. Data were extracted from the scanned image using Feature Extraction V11.5.1.1 (Agilent Technologies, Redwood City, CA, USA) and analyzed using GeneSpring v.7.2 software (Agilent Technologies). Differential translation analysis was assessed with ANOTA analysis as described previously (16 (link)).