DNA isolation method is a source of global DNA methylation variability measured with LUMA. Experimental analysis and a systematic review

PLoS One. 2013 Apr 9;8(4):e60750. doi: 10.1371/journal.pone.0060750. Print 2013.


In DNA methylation, methyl groups are covalently bound to CpG dinucleotides. However, the assumption that methyl groups are not lost during routine DNA extraction has not been empirically tested. To avoid nonbiological associations in DNA methylation studies, it is essential to account for potential batch effect bias in the assessment of this epigenetic mechanism. Our purpose was to determine if the DNA isolation method is an independent source of variability in methylation status. We quantified Global DNA Methylation (GDM) by luminometric methylation assay (LUMA), comparing the results from 3 different DNA isolation methods. In the controlled analysis (n = 9), GDM differed slightly for the same individual depending on extraction method. In the population analysis (n = 580) there were significant differences in GDM between the 3 DNA isolation methods (medians, 78.1%, 76.5% and 75.1%; p<0.001). A systematic review of published data from LUMA GDM studies that specify DNA extraction methods is concordant with our findings. DNA isolation method is a source of GDM variability measured with LUMA. To avoid possible bias, the method used should be reported and taken into account in future DNA methylation studies.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review
  • Systematic Review

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Case-Control Studies
  • CpG Islands
  • DNA / isolation & purification*
  • DNA Methylation / genetics*
  • Diabetes Mellitus / genetics
  • Diabetes Mellitus / metabolism
  • Epigenesis, Genetic*
  • Female
  • Humans
  • Hyperlipidemias / genetics
  • Hyperlipidemias / metabolism
  • Hypertension / genetics
  • Hypertension / metabolism
  • Leukocytes, Mononuclear / metabolism*
  • Luminescent Measurements
  • Male
  • Middle Aged
  • Reproducibility of Results
  • Sensitivity and Specificity


  • DNA

Grants and funding

Grants were awarded by Spain's Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III “Registro BASICMAR” Funding for Research in Health (PI051737) and “GWALA project (PI10/02064)”, and by Spanish Research Networks: “Red HERACLES” (RD06/0009). FEDER. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.