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. 2011 Oct 20;6(10):e26537. doi: 10.1371/journal.pone.0026537

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Beekman et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Overview of the methylation specific PCR (MSP) approach. Depicted is a schematic representation of the Gr1.4 MuLV LTR, containing 23 CpGs. The MSP was performed, after bisulphite treatment, with a methylation neutral forward primer (MN-LTR-fw), and a methylation specific (MS-LTR-rv) or neutral (MN-LTR-rv) reverse primer. Amplification products were quantified using methylation neutral probe-MN. Tumor samples were divided into 4 equal groups based on the methylation status of their LTRs (1 = highest LTR methylation level, 4 = lowest LTR methylation level). (B) Left panel. For H19, the LTR and ActB, average enrichment after MeDIP compared to input levels were calculated for each MSP-defined methylation category as well as for normal bone marrow, spleen and liver (N). In category 1 to 4 respectively 18, 15, 4 and 3 samples were analysed; error bars indicate standard deviations. P-values were calculated using a Wilcoxon test; *significantly higher than other categories, p-value <0.001. Right panel. Example of LTR enrichment after MeDIP (I = input, + = IP with anti-5-methylcytidine, − = IP with pre-immune serum IgG, 1 and 3 = methylation categories, N = normal spleen).