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Abstract
Epigenetic mechanisms, including DNA methylation, are important determinants in development and disease. There is a need for technologies capable of detecting small variations in methylation levels in an accurate and reproducible manner, even if only limited amounts of DNA are available (which is the case in many studies in humans). Quantitative methylation analysis of minute DNA amounts after whole bisulfitome amplification (qMAMBA) has been proposed as an alternative, but this technique has not been adequately standardized and no comparative study against conventional methods has been performed, that includes a wide range of methylation percentages and different target assays. We designed an experiment to compare the performance of qMAMBA and bisulfite-treated genomic (non-amplified) DNA pyrosequencing. Reactions were performed in duplicate for each technique in eight different target genes, using nine artificially constructed DNA samples with methylation levels ranging between 0% and 100% with intervals of 12.5%. Cubic polynomial curves were plotted from the experimental results and the real methylation values and the resulting equation was used to estimate new corrected data points. The use of the cubic regression-based correction benefits the accuracy and the power of discrimination in methylation studies. Additionally, dispersion of the new estimated data around a y = x line (R2) served to fix a cutoff that can discriminate, with a single 9-point curve experiment, whether whole bisulfitome amplification and subsequent qMAMBA can produce accurate methylation results. Finally, even with an optimized reagent kit, DNA samples subjected to whole bisulfitome amplification enhance the preferential amplification of unmethylated alleles, and subtle changes in methylation levels cannot be detected confidently.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
This work was partially funded by Research Project grants from the Instituto de Salud Carlos III of the Spanish Ministry of Economy and Competitiveness (PI10/0310) and from the Basque Department of Industry (SAIO-PE08BF03). NF-J and LP-I are predoctoral fellows supported by FPI grants from the Basque Department of Education, Universities and Research (BIF-2009-099 and BIF-2010-189, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Supplemental Materials
Supplemental materials may be found here: www.landesbioscience.com/journals/epigenetics/article/22846