iHCE cells (passages of 22–23) were cultured in DMEM/Ham’s F12 (1:1) (Gibco, Invitrogen, Paisley, U.K.), 15% FBS (Gibco, Invitrogen), 0.3 mg/ml L-glutamine (Gibco, Invitrogen), 5 µg/ml insulin (Gibco, Invitrogen), 0.1 µg/ml cholera toxin (Calbiochem, La Jolla, CA), 10 ng/ml EGF (Invitrogen, Carlsbad, CA), 0.5% dimethylsulfoxide (DMSO; Sigma, St. Louis, MO), 0.1 mg/ml streptomycin and 1000 IU/ml penicillin (Gibco, Invitrogen). Cells were seeded on collagen coated permeable inserts (Transwell® Polyester Membrane Insert, Costar, Cambridge, MA) and maintained submerged into medium. After one week in culture the medium was supplemented with 40 µg/ml L(+)-ascorbic acid (Sigma, St. Louis, MO) and cells were changed to grow at the air-liquid interface (medium in the apical side was removed). Trans-epithelial electrical resistance (TEER) was followed with an EVOM resistance meter in Endohm chambers (World Precision Instruments, Sarasota, FL).
Total RNA was isolated using TriReagent (Sigma, St. Louis, MO) and RNA samples were further purified using RNAeasy spin columns (Qiagen, Valencia, CA). RNA concentration and purity were determined by measuring absorbances (260 nm and 280 nm) and integrity was determined using the Agilent 2100 BioChip (Agilent Technologies., Palo Alto, CA).
Biotin labeling of cRNA was performed using the ENZO RNA transcript labeling Kit 9 (BioArray High Yeald Labeling Kit, P/N 900182). IVT cRNA cleanup and quantitation, cRNA fragmentation, control targets were performed according to Affymetrix protocols. cDNA was synthesized using T7-(dT)24 Primer (Genset Corp) and Superscript Choice System (Gibco BRL Life technologies).
The hybridization cocktail was made according to Affymetrix protocols. The cocktail was heated to 99°C and then kept on heat block for 5 mins at 45°C. The probe array was incubated with 1X hybridization buffer (MES, 1M [Na], 20mM EDTA, 0.01% Tween) for 10 mins at 45°C. Samples were hybridized for 16 h at 45°C using a final volume of 200 µl. GeneChip Fluidics Station 450 using fluidics protocol EukGE-WS2v4 was utilized for washing. Hybridized microarrays were stained with a streptavidin-phycoerythrin (SAPE) reagent.
Hybridized microarrays were stained with a streptavidin-phycoerythrin (SAPE) reagent and fluorescence images were captured using an Agilent G2500A laser scanner.
Sample comprises of RNA isolated from immortalized human corneal epithelial cells cultured on the collagen coated permeable support at the air-liquid interface.
The data were analyzed with R v. 2.8.0 Bioconductor. Probes present in HGU133A were re-annotated according to Entrez Gene database using custom CDF v. 10. RMA algorithm was used to calculate relative gene expression values.