Absorbance was measured at 570nm using a Multiskan MS ELISA plate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). Laser microdissection Samples were derived selleck chem Rucaparib from surgically removed tissue from six patients with moderately differentiated, Dukes B stage, left-side CRC. In parallel, six adenoma specimens were collected. Paired control non-tumour tissues from patients were obtained from a clinically unaffected site near the resection end and were histologically normal. Tissue samples were immediately frozen in liquid nitrogen after surgery and were stored at ?80��C until the cutting period. Frozen tissue was placed in a cryomold with Tissue Tek embedding medium on dry ice for 1min. Frozen tissue specimens were cut in a series of 6-��m-thick sections onto PALM membrane-mounted glass slides at ?20��C.

After cutting, the slides were taken into dry ice, and were stored at ?80��C until microdissection for up to 48h before staining and dissection. The frozen sections were fixed in ethanol series, and were stained using cresyl violet (Sigma-Aldrich). After staining the tissue, tumour and normal tissues were diagnosed by the pathologist. A total of 5000 epithelial cells were collected from each section using the PALM system (PALM, Bernried, Germany). Microarray analysis Total RNA was extracted from HT29 cells using the RNeasy Mini Kit (Qiagen Inc., Germantown, MD, USA) and from LCM cells using the RNeasy Micro Kit (Qiagen Inc.), according to the manufacturer’s instructions. The quantity and quality of isolated RNA were tested by measuring absorbance and capillary gel electrophoresis using the 2100Bioanalyzer and RNA 6000 Pico Kit (Agilent Inc.

, Santa Clara, CA, USA). Biotinylated cRNA probes were synthesised from 1 to 5��g total RNA and fragmented using the One-Cycle Target Labeling and Control Kit (http://www.affymetrix.com/support/downloads/manuals/expression_s2_manual.pdf), according to the Affymetrix description. In case of LCM samples, two-cycle T7-based linear amplification was performed according to instructions of the manufacturer (Affymetrix Inc., Santa Clara, CA, USA). A volume of 10��g of each fragmented cRNA sample was hybridised into HGU133 Plus2.0 array (Affymetrix) at 45��C for 16h. Slides were washed and stained using Fluidics Station 450 and an antibody amplification staining method according to the manufacturer’s instructions.

Fluorescent signals were detected by a GeneChip Scanner 3000 (Affymetrix). Fifty-three microarrays from colonic biopsy samples (11 normal, 20 villous adenoma, 22 CRC) had been hybridised earlier, their data files were used in a previously published study using different comparisons (Galamb et al, 2008a,2008b, 2009) and are available in the Gene Expession Drug_discovery Omnibus database (series accession numbers: “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).