Scanned images were inspected for artifacts; the percentage of present calls (>25%) and control of RNA degradation were evaluated. Dorsomorphin FDA On the basis of evaluation criteria, all biopsy and HT29 measurements fulfilled the minimal quality requirements. In case of HT29 experiments, the similarity of the 3-3 biological replicates was stated using the Euclidean distance method (Supplementary Figure 1). Affymetrix expression arrays were pre-processed by gcRMA with quantile normalisation and median polish summarisation. Data sets are available in the Gene Expression Omnibus databank for further analysis (http://www.ncbi.nlm.nih.

gov/geo/), series accession numbers: “type”:”entrez-geo”,”attrs”:”extlink”:”1″,”term_id”:”15799″,”text”:”GSE15799″GSE15799, “type”:”entrez-geo”,”attrs”:”extlink”:”1″,”term_id”:”15960″,”text”:”GSE15960″GSE15960, “type”:”entrez-geo”,”attrs”:”extlink”:”1″,”term_id”:”4183″,”text”:”GSE4183″GSE4183 and “type”:”entrez-geo”,”attrs”:”extlink”:”1″,”term_id”:”10714″,”text”:”GSE10714″GSE10714). Further analyses To identify differentially expressed features, significance analysis of microarrays (SAM) was used. The nearest shrunken centroid method (prediction analysis of microarrays=PAM) was applied for sample classification from gene expression data. Prediction analysis of microarrays uses soft thresholding to produce a shrunken centroid, which allows the selection of characteristic genes with high predictive potential (Tibshirani et al, 2002). Pre-processing, data mining and statistical steps were performed using R-environment with Bioconductor libraries.

Annotation and functional classification of discriminatory genes were performed using the Affymetrix NetAffx system. Taqman RT�CPCR TaqMan real-time PCR was used to measure the expression of 12 selected genes using an Applied Biosystems Micro Fluidic Card System (Applied Biosystems, Anacetrapib Foster City, CA, USA). The selected genes belonged to the PAM discriminatory genes between CRC and normal, and between adenoma and normal samples, and validated Taqman assays were available. The following commercially available Taqman Gene Expression Assays (Applied Biosystems) were applied: ABCA8 (Hs00200350_m1), TRPM6 (Hs00214306_m1), VWF (Hs00169795_m1), IL8 (Hs00174103_m1), LCN2 (Hs00194353_m1), CXCL1 (Hs00236937_m1), COL4A1 (Hs00266237_m1), MCAM (Hs00174838_m1), IL1RN (Hs00277299_m1), CXCL2 (Hs00236966_m1), DUOX2 (Hs00204187_m1) and SPP1 (Hs00167093_m1). Ribosomal RNA 18S (Hs99999901_s1) was used as reference. Using the Taqman Reverse Transcription Kit, 400ng per sample of total RNA was reverse transcribed (Applied Biosystems). The quality of cDNA samples was checked by CK20/PBGD real-time PCR (F. Hoffmann-La Roche Ltd., Basel, Switzerland).