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Sci Rep. 2016 Sep 27;6:33936. doi: 10.1038/srep33936.

Experimental factors affecting the robustness of DNA methylation analysis.

Pharo HD1,2,3,4, Honne H1,2,3, Vedeld HM1,2,3, Dahl C5, Andresen K1,2,3, Liestøl K3,6, Jeanmougin M1,2,3, Guldberg P5, Lind GE1,2,3,4.

Author information

Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital, the Norwegian Radium Hospital, Oslo, Norway.
KG Jebsen Colorectal Cancer Research Centre, Oslo University Hospital, Oslo, Norway.
Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
Department of Biosciences, The Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway.
Danish Cancer Society Research Center, Copenhagen, Denmark.
Department of Informatics, University of Oslo, Oslo, Norway.


Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0-100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data.

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