Microarray Analysis of Oral Mucosa

The whole study design, including RNA extraction, microarray assay, and bioinformation steps were described before [11 (link),12 (link)]. In brief, total RNA was prepared with the miRNeasy Mini Kit (Qiagen, 40724 Hilden, Germany). For microarray analysis, we used the Agilent Array platform employing the manufacturer’s standard protocols for sample preparation and microarray hybridization. Gene expression analysis was performed with the Whole Human Gene Expression Microarray (4 × 44K; GPL4133), arrays were scanted with the Agilent G2505B Microarray Scanner and feature extraction was performed with Feature Extraction software version 9.5 (all Agilent Technologies, 76337 Waldbronn, Germany). Data files from mRNA microarrays were analyzed by GeneSpring GX 7.3.1 according to the manufacturer’s protocol (Agilent Technologies, 76337 Waldbronn, Germany). The first normalization step consisted of background elimination while, in a second step, the 50th percentile of each spot was normalized. Normalizations to a healthy oral mucosa pool was performed in the last step with the expression factor for the healthy oral mucosa pool set to 1; the fold change to control is presented in the tables. Primary statistical analysis was performed with GeneSpring GX 7.3.1 software (Agilent Technologies, 76337 Waldbronn, Germany).