The Caco-2 cell line is derived from a male Caucasian with a colon tumor
Biomaterial provider
American Type Culture Collection (ATCC)
Treatment protocol
Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) heat inactivated mycoplasma tested fetal calf serum, 2 mM L-glutamine (6 mM final concentration), 1% (v/v) MEM non-essential amino acids and 50 µg/ml gentamicin. As quercetin in DMEM culture medium was stabilized by 1 mM ascorbate, control cells were exposed to 1 mM ascorbate-only
Growth protocol
Caco-2 cells of passage 37 were seeded (1 : 10 split ratio) in triplicate on polycarbonate membrane transwell® inserts (Corning Life Sciences, Cambridge, U.K.), with a membrane diameter of 75 mm (growth area 44 cm2, 0.4 µm pore size) at a density of approx. 40,000 cells/cm2. After 2 days, cell cultures reached confluency, and this time point was considered experimental day 0 and the initial exposure to quercetin. Cells were exposed for a total number of 9 days
Extracted molecule
total RNA
Extraction protocol
On days 5 and 10 post-confluency, cells were first rinsed with ice cold phosphate buffered saline without calcium or magnesium (InvitrogenTM Life Technologies, Breda, The Netherlands). Subsequently, cells were harvested in 2 x 0.75 ml of ice-cold TRIzol (Life Technologies, Paisley, U.K.), immediately frozen in liquid nitrogen and stored at -80°C for up to 2 months, until RNA isolation according to the TRIzol protocol. Following isolation, total RNA was purified with RNeasy Midi columns (QIAGEN, Westburg, Leusden, The Netherlands), including a DNAse incubation step. Quality of purified total RNA was determined on a UV-VIS spectrofotometer (Shimadzu Benelux, ‘s Hertogenbosch, The Netherlands) by calculation of the A260/A280 ratio (1.7 - 2.0) and confirmed on a Lab-on-a-Chip on the Agilent 2100 bioanalyser (Agilent Technologies, Palo Alto, California, U.S.A.) by determining the 28S/18S ribosomal RNA ratio
Label
Biotin
Label protocol
Labeling protocol according to Affymetrix® GeneChip® Expression Analysis Technical Manual
Hybridization protocol
Hybridization protocol according to Affymetrix® GeneChip® Expression Analysis Technical Manual
Scan protocol
Scan protocol with GeneChip® Scanner 3000 7G, according to Affymetrix® GeneChip® Expression Analysis Technical Manual
Description
Probe sets with an “absent call” (P ≥ 0.065 for HG-U133A 2.0 GeneChip® arrays) across all 8 arrays (n = 7,285) were considered not to be affected by quercetin treatment and/or incubation time and were therefore excluded from further analyses
Data processing
CEL files were imported in Rosetta Resolver 5.0 (Rosetta Inpharmatics LLC, Seattle, U.S.A.), applying data pre-processing (to estimate and reduce systematic errors) and error modeling (for improvement of accuracy of the present and absent calls for low replicate numbers).
