Male Sprague Dawley rat, 40 weeks post-AOM, life-time ingestion of casein based diet (CAS), proximal colon
TriTRIzol extraction procedure, followed by a cleanup using QIAGEN RNeasy Mini kit.
Pooled RNA (equal amounts of RNA from each of 7 animals; 8 ug total) was used for cDNA synthesis using a T7-(deoxythymidine)24 primer and Superscript II (Life Technologies, Inc., Gaithersburg, MD). The resulting cDNA was used with the ENZO BioArray High Yield RNA Transcript labeling kit (ENZO, Farmingdale, NY) to synthesize biotin-labeled cRNA. The cRNA was purified on RNeasy spin columns (QIAGEN) and subjected to chemical fragmentation (size range of 35 to 200 bp). Three replicate cRNA targets were made in parallel starting from each RNA pool.
Ten ug of cRNA was hybridized for 16 hours to an Affymetrix (Santa Clara, CA) rat U34A GeneChip (3 chips used per diet group), followed by incubations with streptavidin-conjugated phycoerythrin, and then with polyclonal anti-streptavidin antibody coupled to phycoerythrin.
GeneChips were scanned using an Agilent GeneArray laser scanner. Images were analyzed using Affymetrix MAS 5.0 software.
Bacterial sequence-derived probes on the arrays served as external controls for hybridization, whereas the housekeeping genes β-actin and GAPDH served as endogenous controls and for monitoring the quality of the RNA target.
To compare array data between GeneChips, we scaled the average of the fluorescent intensities of all probes on each array to a constant target intensity of 500.