Figure 2.
DNA methylation by clonal bisulfite sequencing and by Methyl-Profiler PCR array in a subset of the same sample. Shown are mean ± SE. * p<0.05.
A: Clonal bisulfite sequencing of the CYP1B1 gene as an example of 3 genes in cases and controls (n=5 for each): selected genomic regions were analyzed (208-bp, 28 CpG sites, +733/+940 to mRNA 5′ end, 2p22.2, Genbank accession number NM_000104). PCR amplification of MethylScreen shown in black bar within exon 2 (pink bar). Half arrowheads indicate the PCR primer pair used for Na bisulfate modification-cloning-sequencing. Each row represents an individual clone from the post-bisulfite PCR product. Each column represents a CpG site. Filled circles (in red) indicated methylated CpG sites.
B: Percent of methylated CpG sites in all 3 genes (CYP1B1, GADD45A and CXCL12) in cases and controls (n=5 for each) by clonal bisulfite sequencing. The methylated % was calculated separately from each clone and the average total number of clones for cases and controls were plotted in the histogram graph panel.
C: DNA methylation at 3rd trimester by Methyl-Profiler PCR array analysis in the same subjects of 3 genes.