Estimating Lymphocyte Counts from DNA Methylation

qPCR-based quantitative analysis for single-base methylation (QASM) The methylation percentage of each candidate CpG site was determined in multiple cohorts using a MethyLight-based QASM assay that has been developed and validated in our previous work. In short, the bisulfite-converted DNA was amplified, in which we exploited the locus-specific PCR primers flanking a pair of methylated and unmethylated probes labeled with the fluorescent dyes 6-carboxyfluorescein (6-FAM) and 2-chloro-7phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), respectively. The methylation percentage was calculated by methylation/(methylation+unmethylation)×100%. QASM was performed using the Applied Biosystems QuantStudio 7 Flex Real-Time PCR System (Thermo).