A complementary method was used to validate selected differentially methylated genes. A set of genes, chosen with both average-level differences and two consecutive probe-level differences, were validated using Sequenom MASSarray. PCR primers were designed to assay 26 gene promoter regions with 48 amplicons covering 9 CpGs per amplicon. Each primer pair included a T7 promoter sequence. These primers were used to amplify bisulfite-treated genomic DNA. Following in vitro transcription, and nucleotide-specific RNA cleavage (MassCLEAVE), MALDI-TOF mass spectrometry was used to acquire spectral peaks to create methylation ratios based on the number of C residues representing methylated CpGs or U residues due to bisulfite-conversion of unmethylated CpGs. Genes were defined as having a methylation difference if they had greater than 10% of at least one CpG unit altered and changes in the same direction for all CpG units within an amplicon. The Sequenom protocol validated 12 genes of the 26 predicted to be differentially methylated by 5mC-immunoprecipitation and microarray. Overall, verified hypermethylation was found in genes such as insulin growth factor (Igf2), SRY-box containing gene 7(Sox7), and cut-like 2 (Cux2) that
