Global DNA (hydroxy)methylation is stable over time under several storage conditions and temperatures

Background: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation. Methods: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4 degrees C, -20 degrees C and -80 degrees C for 0, 1 (endpoint blood 4 degrees C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean +/- SD) were quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models. Results: global DNA methylation was stable over 18 months in blood at -20 degrees C and -80 degrees C and DNA at 4 degrees C and -80 degrees C. However, at 18 months DNA methylation from DNA stored at -20 degrees C relatively decreased -6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze - thaw cycles. Conclusion: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of small differences occuring during storage depend on the expected effect size and research question.

Automated summary

This study examined the stability of global DNA (hydroxy)methylation under different storage conditions and found that these markers can be stable for up to 18 months under specific conditions. DNA samples showed more stability in global DNA hydroxymethylation compared to blood, and freeze-thaw cycles did not significantly affect the stability of methylation and hydroxymethylation.