DNA Extraction, Bisulfite Modification and Methylation Profiling (Infinium Methylation Assay)

DNA extraction was performed with the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. Genomic DNA was bisulfite modified (EZ DNA methylation Gold Kit, Zymo Research, please add city/state) as described previously [35]. To analyze the effect of curcumin on DNA methylation, we performed DNA methylation profiling using the Infinium methylation assay with HumanMethylation27 BeadChip microarrays (Illumina, San Diego, CA), which are capable of simultaneously analyzing the methylation status of 27,578 individual CpG sites covering over 14,000 genes [36]. Whole genome amplification, labeling, hybridization and scanning were performed according to the manufacturer’s instruction at the genomics core facility at the Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX. Methylation status was measured as the ratio of signal from a methylated probe relative to both methylated and unmethylated probe signals. Methylation ratios were extracted using the Methylation Modules in the Illumina Bead Studio following average normalization, and the quantitative β-value ranged from 0 (0% methylation) to 1 (100% methylation). The p-value cut-off for detectable probe signals was set at 0.05. For the methylation analyses, a Δβ-value of ≥0.1 (10% methylation) was defined as significant [37].