At confluence, keratinocytes from the DK-7 cell line were exposed to 100 IU/ml of IFN-g in serum free medium. After 24 hrs, RNA was extracted using the Qiagen Rneasy Mini Kit (Qiagen SA, Cedex, France). All the samples were monitored by agarose gel and with the Agilent 2100 Bioanalyser (Agilent Biotechnologies, Germany) and consistently demonstrated high-quality RNA (28S/18S ratio approximately 2, but always less than 3). According to Affymetrix protocol, 5 µg total RNA was the starting material for all individual samples. In general, total RNA was converted to biotinylated cRNA, hybridized in the Affymetrix probe array cartridge, stained, and then quantified. First and second strand cDNA synthesis was performed using the SuperScript Choice System (Invitrogen AG, Basel, Switzerland), according to manufacturer instructions, but using an oligo-dT primer containing a T7 RNA polymerase binding site. Labeled cRNA was prepared with the RNA Transcript Labeling kit (Enzo Biochem Inc., NY). Biotinylated CTP and UTP were used together with unlabeled NTPs in the reaction, and unincorporated nucleotides were removed with GeneChipâ Cleanup Module (Affymetrix, Inc., Santa Clara, CA) . cRNA (20 µg) was fragmented at 94 °C for 35 min in buffer containing 200 mM Tris-acetate pH 8.1, 500 mM KOAc, and 150 mM MgOAc. Prior to hybridization, fragmented cRNA in hybridization mix ( Buffer containing 100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20, 0.5 ng/ml BSA, 0.1 ng/ml herring sperm and Affymetrix controls ), was heated to 95 °C for 5 min, cooled to 45 °C and loaded onto an Affymetrix probe array cartridge. The probe array was incubated for 16 hrs at 45 °C at constant rotation (60 rpm), then exposed to Affymetrix washing and staining protocol. This protocol includes: · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) · one wash with stringent buffer ( 100 mM MES, 0.1 M NaCl, 0.01 % Tween 20 ) · First stain with 0.01 mg/ml streptavidin-phycoerythrin conjugate (Molecular Probes) in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) · Second stain with 3 mg/ml of biotinylated anti-streptavidin + 0.2 mg/ml of IgG in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · Third stain with 0.01 mg/ml streptavidin-phycoerythrin conjugate (Molecular Probes) in buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20, 4 mg/ml of BSA. · one wash with non-stringent buffer ( 6X SSPE, 0.01% Tween 20, 0,005% antifoam ) Probe arrays were scanned at 488 nm using an Argon-ion Laser (made for Affymetrix by Agilent). Readings from the quantitative scanning were analyzed with Affymetrix Gene Expression Analysis Software (MAS 5.0). This experiment was comparing control versus IFN-g treated skin cells (one design factor on two levels). To evaluate the biological and the experimental variability, the following experimental design was planned as shown in Figure 1: "Control" and "IFN-g stimulated" keratinocytes were cultured in triplicate using three individual petri Æ10 culture dishes and RNA was independently extracted from each cultured replicate. For each RNA sample, cRNA synthesis was performed in triplicate and each cRNA pool was hybridized to an Affymetrix U133 Gene Chip.
