Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR

Abstract

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context-density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.

Quantification of Unmethylated Insulin DNA Using Methylation Sensitive Restriction Enzyme Digital Polymerase Chain Reaction

“…Based on these notions, we suggest in this preliminary study that our newly developed MSRE duplex digital PCR assay using unmethylated INS -DNA may be a fast and easy method to specifically quantify β-cells. As shown previously, the combination of MSRE with digital PCR provides both specificity and sensitivity by quantitative assessment of target alleles ( 14 ). By measuring the concentration of the targeted unmethylated INS -DNA and therefore the number of lysed β-cells, this combined technique may be a promising tool to determine the fraction of β-cells immediately after islet isolation, during culture and immediately prior to islet transplantation.…”

“…To avoid bisulfite conversion whilst still allowing the possibility to specifically quantify the methylation fraction of a specific allele, we recently published a methylation sensitive restriction enzyme (MSRE) digital PCR assay ( 14 ). MSREs are used to differentiate between methylated and unmethylated alleles and in combination with digital PCR it provides the opportunity to determine specific allele quantification.…”

WITHDRAWN: The epigenetic study of Fgfr2 as a potential biomarker in the differential diagnosis of intestinal metaplasia and gastric cancer

“…In the ideal state, this conversion deaminates unmethylated cytosines to uracil but does not affect methylated cytosines. In the experimental condition, DNA manipulation and treatments may result in incomplete conversion, falsepositive extensive, and DNA degradation (of up to 90%) [20]. Because the WBCs extracted DNA's amount and quality are high, studies are shifting to assay WBCs DNA as a potential biomarker [21].…”

Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples Using Multiplex Digital PCR

“…The key elements of digital PCR (subsampling, random partitioning, end-point measurements, and consequent mathematical interpretation) can be effectively modeled in silico, providing important insights in the mathematical validity and precision of a given experimental setup. 10,21 This analysis demonstrated that under copy number stable conditions, the T-cell fraction is correctly estimated by using the adjusted model, but with slightly decreased precision than in the classic model (Figure 3A). These findings were confirmed by the analysis of healthy PBMC DNA samples using both the classic and adjusted model (Figure 5A).…”