Comparative Analysis of Two Digital PCR Platforms for Detecting DNA Methylation in Patient Samples

Abstract

The choice of appropriate analytical methods for determining methylation patterns at specific loci across the genome is essential for identifying novel diagnostic and prognostic markers for subsequent clinical implementation. Various methods exist for determining methylation status using different technologies. In this study, we compared two distinct digital polymerase chain reaction (PCR) platforms: the nanoplate-based Qiagen QIAcuity Digital PCR (dPCR) System and the droplet-based Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. By assessing their efficacy and other attributes, we aimed to elucidate each platform's strengths and limitations in the sensitive detection of DNA methylation, thus contributing valuable insights to the field of molecular diagnostics. We analyzed the methylation status of the CDH13 gene in 141 formalin-fixed, paraffin-embedded breast cancer tissue samples using our in-house developed methylation-specific labeled assay. The specificity and sensitivity of the CDH13 assay evaluated by dPCR were 99.62% and 99.08%, respectively; ddPCR analysis reached a specificity of 100% and a sensitivity of 98.03%. In addition, our data revealed a strong correlation between the methylation levels measured by both methods (r = 0.954). Although both methods are based on different technologies, they yielded comparable, highly sensitive experimental data in our study. Consequently, the main criteria for selecting an optimal digital PCR platform for methylation analysis may lie in other factors such as workflow time and complexity, instrument requirements, the possibility of temperature gradient, reanalysis, or offline options.