2.4. RNA Extraction and Microarray Hybridization

Total RNA was extracted from treated NHDF cells using Qiazol (Qiagen, Valencia, CA, USA) reagent followed by a clean-up treatment with the RNeasy Mini kit (Qiagen) according to manufacturer’s recommendations to remove contaminating genomic DNA. RNA integrity and purity were assessed by Bioanalyzer (Agilent Technologies, Waldbronn, Germany) and concentration was evaluated using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA).

RNA samples were processed for microarray hybridization by INT Functional Genomics core facility. Briefly, 300 ng of total RNA were reverse-transcribed, labeled with biotin and amplified overnight with the Illumina RNA TotalPrep Amplification kit (Ambion, Austin, TX, USA) according to manufacturer’s instructions. One µg of biotinylated cRNA was mixed with the Hyb E1 hybridizatioin buffer containing 37.5% (w/w) formamide and then hybridized to Illumina HumanHT-12v4 Expression BeadChip (Illumina, Inc., San Diego, CA, USA) at 58 °C for 18 h. Arrays were washed with manufacturer’s E1BC solution (Illumina Inc, San Diego, CA, USA), stained with 1 µg/mL Cy3-streptavidine (GE Healthcare, Buckinghamshire, UK) and scanned with Illumina BeadArray Reader. Illumina BeadScan software was used for image acquisition and recovery of primary signals.