© 2012 Landes Bioscience. Do not distribute. Figure 1. s chematic of analyses performed. Analyses of element age were conducted in two tissue groups: DNA methylation of REs is dependent on both evolutionary age (1) merged villi (placental villi from all three gestational ages) and and assay method. (2) merged somatic tissues (fetal brain, fetal kidney, fetal muscle Evidence for association of weak island probe methylation and adult blood), since DNA methylation of each subfamily of with LINE-1 and non-island probe methylation. Correlation Alu or LINE-1 did not differ between the tissues included in analyses were performed to determine how LINE-1 and Alu each of these groups (data not shown). There was a significant DNA methylation assessed by Pyrosequencing compare with trend for hypomethylation of older LINE-1 and Alu subfamilies each other and to DNA methylation of other ReDS. Using aver- in comparison to intermediate and young in both the merged age DNA methylation assessed by the array, samples clustered villi and somatic groups (Fig. 2). We next compared the level of most strongly by placental vs. somatic origin (Fig. S1), and then RE DNA methylation assessed by Pyrosequencing to array probe by tissue type; thus, inter-method comparisons were conducted assessment. After correction for multiple comparisons, mean within each somatic tissue (fetal brain, kidney and muscle and LINE-1 methylation by Pyrosequencing was not different from adult blood) and then collectively in the merged somatic group. LINE-1H methylation assessed by array probes in the merged Gestational age was previously found to have a significant effect villi group (Fig. 2B). All other comparisons of DNA methyla- on villi DNA methylation,34,35 therefore, inter-method compari- tion between LINE-1/Alu Pyrosequencing and array probes were sons of villi were performed within each gestational age group. RE significantly different in both tissue groups (Fig. 2A and B, all DNA methylation was compared with three groups of Illumina p < 0.001). Assessment by Pyrosequencing of LINE-1 DNA Infinium HumanMethylation27 BeadChip array probes catego- methylation was correlated with array results in the somatic rized based on CpG island density: strong, weak and non-island tissue group (LINE-1M, r = 0.55; LINE-1P, r = 0.58; both p < probes. Table S1 summarizes average DNA methylation by tis- 0.0001) but in neither tissue group for Alu methylation. Thus, sue group for each of the five ReDS. There were no significant 654 Epigenetics Volume 7 Issue 6