mRNA levels that were inversely correlated with promoter methylation (.40–60% of CpG sites) in 35–59% of melanoma cell lines and 6–25% of the fresh tumors. In order to identify additional epigenetically silenced genes implicated in melanocytic neoplasia, we have generated new data from 11 melanoma cell lines using Illumina Infinium Methyla- tion27 arrays [17]. In order to provide a more complete picture of methylation in melanoma, these data were integrated with our previous constitutive mRNA expression [18,19] and post-demeth- ylation treatment expression data [14] to identify a list of potential genes for assessment by comprehensive promoter methylation analysis using the Epityper system in an extended panel of melanoma cell lines and tumors. Results Description of a new pipeline to identify novel candidate gene CpG islands methylated in melanoma In our previous study, we used a microarray-based strategy in a panel of 12 melanoma cell lines treated with 5AzadC and TSA as an initial screening approach. Select candidate genes were followed up using the Epityper assay in a much larger panel of melanoma cell lines, as well as a panel of fresh-frozen melanoma samples, normal melanocyte cultures, and cell lines from other cancer types. We identified four genes, PPP1R3C, ENC1, RARRES1 and TP53INP1 Epigenetic Gene Silencing in Human Melanoma that were not previously known to be silenced by DNA methylation in melanoma [14]. In order to use more robust criteria to select the genes epigenetically silenced during the development of melanocytic tumors, we have generated new data on 11 melanoma cell lines using Illumina Infinium Methylation arrays. The Infinium Methylation chips interrogate 27,578 CpG loci covering more than 14,000 genes. We screened 11 melanoma cell lines from our pilot study [14], which we compared to pools of melanocytes from several donors. These new data were then integrated with previous data-sets of global mRNA expression [18,19] and expression post-demethylation treatment [14] in order to focus on identifying additional candidate TSGs down-regulated through promoter methylation. Genes were further filtered to identify those in which $60% methylation correlated with a 4-fold decrease in mRNA levels in at least 2 samples, together with an average post-demethylation re-expression fold-change of .4 across the panel of 11 melanoma lines (Figure 1). This gave a set of 26 genes, from which we then removed oncogenes (ADM, ENPP2, RAC2, SERPINE1), genes we had previously identified (PPP1R3C), those without a described function e.g. annotated as ‘‘orf’’ (C10orf116, SLC25A38, CCDC109B), and false positives on the Infinium Methylation chips (i.e. multiple instances where cell lines were methylated on the HumanMethylation27 BeadChip but showed no re-expression with 5AzadC+TSA treatment; EEF1A2, HSPA2). Figure 1. Flow through of the cross-platform array integration. doi:10.1371/journal.pone.0026121.g001 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26121