Analysis of CRY1 CpG island promoter methylation status measured by bisulphite genomic sequencing.

<p><b>A</b> Results of bisulphite genomic sequencing performed on 6–12 clones of 4 normal donors (ND) and 12 different CLL samples. Top, schematic depiction of the analyzed area of the CRY1 CpG island around the transcription start site (horizontal black line). Short vertical lines represent CpG dinucleotides. The location of PCR primers are indicated as bold black horizontal arrows. Presence of methylated or unmethylated CpG dinucleotides is indicated by solid or open squares respectively, blue squares indicate not analyzable CpG dinucleotides. The mean standard error for all samples is 1.99 reflecting the low variability between the methylation profile of each analyzed clone. At the right hand side of each graph, first the percentage of methylated CpGs for each individual sample is depicted, below is the percentage of methylated CpGs measured by Bisulphite MassArray and at the bottom each sample's CRY1 mRNA expression relative to GAPDH is shown. <b>B</b> Samples from CLL patients and ND were subjected to both CRY1 mRNA expression and DNA methylation analysis by bisulphite genomic sequencing. mRNA expression values and percentage of methylated CpG were found to be highly correlated (r = −0.62, p = 0.006, Spearman correlation). The regression line in the plot was produced by linear regression analysis using promoter methylation as dependent and CRY1 mRNA expression as independent variable (open circles indicate ND).