Gene Expression Microarray Analyses

In parallel with methylation profiling, we performed microarray gene expression analyses following the manufacturer’s instructions and a previously established protocol [39]. Total RNA was isolated using the RNeasy mini-kit (QIAGEN, Valencia, CA) and amplified using Illumina’s TotalPrep RNA Amplification Kit. RNA integrity was assessed using the Agilent 2100 Bioanalyzer. Labeled cRNA was hybridized overnight to Human HT12 V3 chips, washed, and scanned on an Illumina BeadStation-500. Illumina’s BeadStudio version 3.1 was used to generate signal intensity values from the scans, subtract background, and scale each microarray to the median average intensity for all samples (per-chip normalization). Normalization was done using quantiles with the Lumi R-package. Fold-changes were calculated with respect to their respective controls as described elsewhere [35]. Ingenuity pathway analysis (IPA, Ingenuity Systems, Inc. CA, USA) was performed to evaluate und categorize differentially expressed genes into various functional pathways.