Fig. 3

Spike and recovery test of the ddMSP detection system and validation using samples of patients with mesothelioma. A Human serum was spiked with pre-determined copies of ccfDNA from H28 mesothelioma cells or fully methylated genomic DNA, and ccfDNA was purified following bisulfite conversion. The copy numbers of methylated DKK3 were quantified via ddPCR. B Typical scatter plots obtained from the experiments are shown. The X-axis shows the number of copies spiked into human serum; the Y-axis shows the number of copies recovered after the process. From each point, a linear regression curve was drawn in each bisulfite conversion. C Typical scatter plots obtained from the experiments. The Y-axis shows the percentage of methylated DKK3 copies per unmethylated DKK3 derived from human serum. Assays were performed in two independent experiments, and the representative one is shown. Determination of the methylation status in the DKK3 promoter region of ccfDNA in patients with malignant mesothelioma. D Patients with mesothelioma were categorized based on stage; the y-axis shows the number of methylated copies per 4 mL of serum. E Patients with mesothelioma were classified based on disease type, and the number of methylated copies for each disease type is shown on the Y-axis. The mean value for each population is indicated as a bar. ddMSP droplet digital methylation-specific polymerase chain reaction; ccfDNA circulating cell-free DNA; DKK3 Dickkopf-related protein