Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

Yoonsoo Hahn,Hannah Yu,Osman El-Maarri,Inchul Yang

doi:10.1371/journal.pone.0137006

Abstract

Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.

Highlights

Cytosine methylation at CpG sites plays pivotal roles in gene expression regulation and the maintenance of cellular functions in vertebrates [1,2]
Inconsistent receiver operating characteristic (ROC) data resulting from variation in measurement sensitivity and specificity limits the effectiveness of DNA methylation-based diagnostics [7,8]
We developed reference materials to examine and calibrate PCR biases in DNA methylation analyses

Summary

Introduction

Cytosine methylation at CpG sites plays pivotal roles in gene expression regulation and the maintenance of cellular functions in vertebrates [1,2]. Use of reference materials of which DNA methylation values were accurately determined could be a solution for precise verification and for appropriate calibration of PCR biases in DNA methylation analyses. Pairs of reference materials of which DNA methylation levels and relative concentrations are accurately known with respect to specific genes could provide improved solutions for correct verification and calibration of PCR biases in the quantification of DNA methylation.

Results

Conclusion