Fig. 2

Confirmation of specificity for methylated and unmethylated templates in ddPCR and validation in mesothelioma cell line. A Gradient PCR was performed within an annealing temperature range of 50–61 °C with the matched or mismatched template. U bisulfited-fully unmethylated genome and M bisulfited-fully methylated genome. B Each PCR product was subjected to electrophoresis. C Changes in the amount of ccfDNA after bisulfite treatment using each kit. Selection of bisulfite conversion kit for ccfDNA. The X-axis shows the ccfDNA input (the amount of DNA before bisulfite conversion), and Y-axis shows the number of recovered ccfDNA (the amount of DNA after bisulfite conversion). The experiments were performed in duplicate, and each mean value was plotted and fitted into a linear regression model. The copy number of methylated DKK3 in mesothelioma cell lines, mesothelial cells, and non-cancer ccfDNA was determined. D The methylation status of DKK3 is shown based on analysis of reduced representation bisulfite sequencing (https://depmap.org/portal/). E Copy number of methylated and unmethylated DKK3 in human mesothelioma cell lines (MSTO-211H, NCI-H28, NCI-H226, and NCI-H2052), mesothelial cell line (Met5A), and non-cancer cell-free DNA (derived from 2 donors). ddPCR droplet digital polymerase chain reaction; ccfDNA circulating cell-free DNA; DKK3 Dickkopf-related protein