Mucosal biopsies were obtained from patients with chronic inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn’s disease (CD) diagnosed according to generally accepted standard (Lennard-Jones PMID 2617184). Consecutively included were IBD patients followed at the IBD clinic Northsealand Hospital and referred to colonoscopy or sigmoidoscopy due to clinical activity of at least 14 days duration. As control patients were included persons referred to endoscopy for reasons other than IBD revealing normal endoscopy. Exclusion criteria were for IBD patients and controls: age below 18 years, any gastrointestinal disease other than IBD, malignant disease, or known autoimmune disease not associated to IBD. Totally 109 biopsy donors were included, 53 UC, 25 CD, and 28 controls.
Growth protocol
Human samples, no growth protocol
Extracted molecule
polyA RNA
Extraction protocol
All intestinal biopsies were placed immediately in RNAlater (Qiagen) stored in a refrigerator and later transferred to a -80ºC freezer for long time storage until RNA extraction. The biopsy tissue was transferred into a tube and homogenized with Ultra Turrax (IKA, Staufen, Germany), together with 700µl Qiazol reagent (company ref). The quality and concentration of the purified total RNA was investigated on a Bioanalyzer 2100 (Agilent) using the RNA 6000 Nano Labchip kit (Agilent technologies, Santa Clara, CA), and all RNA sample concentrations were measured on a ND-8000 NanoDrop (Thermo Fisher Scientific, Waltham, MA). RNA quality was evaluated based on the 28S/18S ratio RIN (RNA integrity number. The RIN value from the human biopsy materials varied but all values above 7 were used for Array. RNA was stored at -80°C until use.
Label
biotin
Label protocol
RNA was hybridized to Affymetrix GeneChip HT HG-U219 arrays, and processed in a Gene Titan Platform (Affymetrix, Santa Clara, USA).
Hybridization protocol
RNA was hybridized to Affymetrix GeneChip HT HG-U219 arrays, and processed in a Gene Titan Platform (Affymetrix, Santa Clara, USA).
Scan protocol
RNA was hybridized to Affymetrix GeneChip HT HG-U219 arrays, and processed in a Gene Titan Platform (Affymetrix, Santa Clara, USA).
Data processing
The resulting CEL files were pre-processed using the RMA algorithm as implemented in Affymetrix Power Tools, including a log2 transformation step (version 1.16.1) [cite 12925520]. The probes were summarized using custom CDF files remapped to Ensembl genes (Brain array) [PMID 16284200].
