Cell monolayers were treated with 100 pg/ml CXCL8, added to the cells diluted in culture medium. After 6 hours of incubation, Caco-2 cells were incubated with TriZol reagens until lysis was complete, then RNA extraction and purification were performed
Growth protocol
Caco-2 cells were grown in DMEM (Invitrogen) containing Glutamax and supplemented with 10% heat-inactivated fetal bovine serum (PAA laboratories, Colbe, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma, St. Louis, MO) in 6-wells microtiter plates (Costar) until polarised (14 days), maintained at 37°C in a humidified 5% CO2/95% O2 atmosphere.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from Caco-2 cell line samples with TRIzol reagent, purified and DNAse treated using the RNeasy mini kit (Qiagen, Venlo, the Netherlands) and DNase (Qiagen, Venlo, the Netherlands) following the manufacturer’s instructions. RNA integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands) with 6000 Nano Chips according to the manufacturer’s instructions. RNA was judged as suitable for array hybridization only if samples showed intact bands corresponding to the 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RIN (RNA integrity number) above 8.0.
Label
biotin
Label protocol
Total RNA (500 ng) extracted fromCaco-2 cells was labelled using the GeneChip 3’ IVT Express kit (cat. no. 901229).
Hybridization protocol
The labelled RNA was hybridised to a GeneChip Hu133 Plus2 array (Affymetrix, Santa Clara, CA), washed, and stained. Detailed methods for the labelling and subsequent hybridisations to the arrays are described in the eukaryotic section of the GeneChip Expression Analysis Technical Manual, Revision 3, from Affymetrix.
Scan protocol
The GeneChip Hu133 Plus2 array was scanned on an Affymetrix GeneChip 3000 7G scanner. Detailed methods are described in the eukaryotic section of the GeneChip Expression Analysis Technical Manual, Revision 3, from Affymetrix
Data processing
Packages from the Bioconductor project, integrated in an in-house developed on-line management and analysis database for multiplatform microarray experiments, were used for analysing the scanned Affymetrix arrays (Gentleman et al., 2004; Gavai et al, submitted).
