title: Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.
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.
authors.affiliation: Center for Bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science, Daejeon, 305-340, Republic of Korea; Bio-analytical Science, University of Science and Technology, Daejeon, 305-340, Republic of Korea.
authors.forename: Hannah
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, 156-756, Republic of Korea.
authors.forename: Yoonsoo
authors.initials: Y
authors.lastname: Hahn
authors.affiliation: Center for Bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science, Daejeon, 305-340, Republic of Korea; Bio-analytical Science, University of Science and Technology, Daejeon, 305-340, Republic of Korea.
authors.forename: Inchul
authors.initials: I
authors.lastname: Yang
mesh_terms: D003562:Cytidine / Q000032:analysis; D004247:DNA / Q000032:analysis; D019175:DNA Methylation; D016133:Polymerase Chain Reaction / Q000592:standards*
chemical_list: D003562:Cytidine; D004247:DNA