1 were used for further microarray analysis at the VIB Nucleomics Core (http://www.nucleomics.be). learn more Per sample, an amount of 100 ng of total RNA spiked with bacterial RNA transcript positive controls (Affymetrix) was amplified and labelled using the GeneChip 3′ IVT express kit (Affymetrix). All steps were carried out according to the manufacturer’s protocol. A mixture of purified and fragmented biotinylated aRNA and hybridisation controls (Affymetrix) was click here hybridised on Affymetrix HG U133 Plus 2.0 arrays followed by staining and washing in a GeneChip® fluidics station 450 (Affymetrix) according to the manufacturer’s procedures. To assess the raw probe signal intensities, chips were scanned using a GeneChip® scanner
3000 (Affymetrix). The RMA procedure was used to normalize data within arrays (background correction and log2-transformation) and between arrays (quintile normalization) SGC-CBP30 (affy_1.22.0 package of Bioconductor)
[14, 15]. The MAS 5.0 algorithm (Microarray suite user guide, version 5; Affymetrix 2001) was used to assess detection above background. All probesets had a good signal and were used for further analysis. Four experimental designs were analysed: the effect of PDAC patients with a good outcome (‘Good’) versus surrounding pancreatic tissue (defined as ‘control’), the effect of PDAC patients with a poor outcome (‘Bad’) versus surrounding pancreas, the effect of ‘Bad’ versus ‘Good’ and the effect of all PDAC samples, irrespective of outcome, versus metastatic disease in the liver or peritoneum . The limma package from Bioconductor was used to assess the contrast in each experiment [16]. Statistical significance of this contrast was tested with a moderated t-test ADAMTS5 (implemented in limma). Differentially expressed genes were defined as genes with an uncorrected p-value of p < 0.001 in combination with >2 fold-change. Classical schemes to adjust for multiple testing can result in low statistical power for microarray studies . The stringent cut-off of p < 0.001 was used as an alternative, pragmatic approach to balance the number of false positives and false negatives
[17]. Metastatic samples (LM and PM) were contaminated with respectively normal liver and peritoneal tissue, reflecting in upregulation of liver- and peritoneal specific genes. Therefore only genes that were not differentially expressed between LM and PM samples, considered as metastatic specific genes, were used for analysis between primary tumour and metastatic tissue. All gene expression data will be available from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/projects/geo/). Functional pathway analysis on differentially expressed probe sets was done with the Ingenuity Pathway Analysis (IPA) program (Ingenuity Systems, http://www.ingenuity.com; Redwood City, CA). For each experiment, probe sets with a corrected p-value <0.001 and a >2 fold change were used as input.