subject number: Subject11 disease status: IBD gender: male age: 54 intervention status: before
Treatment protocol
Subjects with a before AND after measurement participated in a 7-day red meat intervention study (300 grams of red meat per day). Subjects with only a before measurement did not participate in any intervention
Extracted molecule
total RNA
Extraction protocol
During a colonoscopic examination six biopsies were taken from mucosal tissue in visually non-inflamed regions of the colon (in most cases from the sigmoid and descending colon) which were immediately frozen in liquid nitrogen and stored at -80 °C until use. Three biopsies were separately dissolved in QIAzol® (Qiagen, Venlo, The Netherlands) using a tissue disruptor and subsequently pooled. Total RNA was subsequently extracted according to the manufacturer's instructions.
Label
Cy3
Label protocol
Dye-labeled cRNA (Cy3) was synthesized following the Agilent one-color Quick-Amp labeling protocol (Agilent Technologies, Amstelveen, The Netherlands).
Hybridization protocol
Hybridization according to Agilent instructions on 4x44K human whole genome microarrays.
Scan protocol
Slides were scanned on a GenePix® 4000B Microarray Scanner (Molecular Devices, Sunnyvale, USA). Cy3 was excited at a wavelengths of 532 nm. Laser power was set to 100%. The photo multiplier tube gain was set to a saturation tolerance of 0.02% to minimize background and saturated spots. Photomultiplier gain was set to 522 (Cy3), based on automatic establishment of ideal scan settings. Images were subsequently saved (resolution 5 micron, 16 bit tiff image).
Description
No intervention
Data processing
Bad and empty spots were flagged using the GenePix Pro software (version 6.0, Molecular Devices, Sunnyvale, CA). Quality control was performed in the statistical software environment R (version 2.10.1, The R Foundation for Statistical Computing, Vienna, Austria). Quantile normalization and subsequent data processing including background subtraction and Log2 transformation of spot intensities was performed in ArrayTrack (version 3.4, NCTR, Jefferson, AR).
