Application of MeKL-chip to identify T-DMRs in the mouse retina and brain. The fold-change between the retina and brain DNA samples was calculated and combined for samples used for MeKL-chip. The mean fold enrichment and standard error of the mean are plotted for triplicate enrichment experiments (three mice, three DNA samples). (A) Post-MBD enrichment, QPCR showed Rbp3 (grey) and Rho (black) were enriched for methylated DNA in the brain samples as expected. Also as expected, no differential enrichment was evident for H19 (white). (B) Post-KLM-PCR, QPCR showed maintenance of the enrichment pattern for methylation in the brain samples for Rho and Rbp3, while the amount of the H19 template remained equal in both tissues. (C) Relative CpG methylation profiles as evaluated using MeKL-chip (top) and pyrosequencing validation (bottom) of the top T-DMR at Rgs20 for the brain (blue, dashed) and retina (red, solid) from 250 ng of starting DNA. Each point is the relative methylation at one probe from one sample; blue and red lines show the average methylation of triplicates. Near the highest ranked T-DMR (black box, P < 10-16) is a second T-DMR (P < 0.0091, dashed box). The alternative TSS of the brain-specific isoform of Rgs20 is indicated by a dashed arrow at Exon 3. Bisulfite pyrosequencing of seven CpGs within Intron 2 confirmed differential methylation between the mean of retina (red bars) and brain (blue bars) in five mice (Student’s two-tailed, paired t-test, P < 0.001). Error bars show the 95% CI (n = 5). KLM-PCR: kinase-modified ligation-mediated PCR; MBD: methyl-binding domain protein; MeKL: MBD2b/MBD3L1 enrichment of DNA followed by KLM-PCR; QPCR: quantitative PCR; T-DMR: tissue-specific differentially methylated region; TSS: transcription start site.