Amplified cDNA targets were prepared using the Nugen WT-Ovation FFPE System v2 in combination with the Nugen FL-Ovation cDNA Biotin Module v2 and were performed in accordance with manufacturer’s instructions.
Microarrays were scanned on a single Affymetrix 7G scanner.
Hybridization, washing, staining and scanning of fragmented, labelled cDNA was carried out according to standard Affymetrix protocols. Between 3.0 and 3.5µg fragmented, labelled cDNA was hybridized to the Colorectal Cancer DSA microarray (Almac Diagnostics), which contains 61,528 probe sets expressed in both Colorectal tumors and normal tissue.
Total RNA was extracted from the macrodissected FFPE tumor samples using the Roche High Pure RNA Paraffin Kit.
RMA normalised CEL intensity data processed using the Affymetrix Power Tools, version 1.14.2, using the 'rma-sketch' analysis pathway. Files of the second batch were quantile-normalised using the distribution of the first batch as a target. Probeset-level signals were there mean-aligned to the observed averages of the stage-3 samples of the first batch by additive correction.