RESEARCH ARTICLE Reference Materials for Calibration of Analytical Biases in Quantification of DNA

Most contemporary methods for the quantification of DNA methylation employ bisulfite con-version and PCR amplification. However, many reports have indicated that bisulfite-medi-ated PCRmethodologies 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 % inter-vals 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 sequenc-ing-based analyses of DNAmethylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent meth-ylation measurement processes, resulting in accurate measurements