ABSTRACT

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°C, −20°C and −80°C for 0, 1 (endpoint blood 4°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°C and −80°C and DNA at 4°C and −80°C. However, at 18 months DNA methylation from DNA stored at −20°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.

摘要

背景：表观遗传标记通常被量化并与储存样本中的疾病相关。同时，尚未彻底检查储存对这些标记物稳定性的影响。在这项纵向研究中，我们调查了储存时间、材料、温度和冻融循环对整体 DNA（羟基）甲基化稳定性的影响。方法：从 90 个人收集 EDTA 血液。血液（n = 30，第 1 组）和提取的 DNA（n = 30，第 2 组）分别在 4°C、-20°C 和 -80°C 下储存 0、1（终点血液 4°C）、6、 12 或 18 个月。此外，血液和 DNA 样本（n = 30，第 3 组）的冻融循环进行了三天。使用液相色谱-电喷雾电离-串联质谱法 (LC-ESI-MS/MS) 量化整体 DNA 甲基化和羟甲基化（平均值±SD），运行间精度为 2.8%（甲基化）和 6.3%（羟甲基化）。使用线性混合模型评估对稳定性的影响。结果：在 -20°C 和 -80°C 的血液以及 4°C 和 -80°C 的 DNA 中，整体 DNA 甲基化在 18 个月内是稳定的。然而，在 18 个月时，与基线相比，储存在 -20°C 下的 DNA 的 DNA 甲基化相对减少了 -6.1%。与血液相比，DNA 样本中的整体 DNA 羟甲基化更稳定，与温度无关（p = 0.0131）。全局 DNA 甲基化和羟甲基化的稳定性在最多三个冻融循环中没有受到影响。结论：以血液和 DNA 形式存储的全局 DNA 甲基化和羟甲基化可用于表观遗传研究。储存期间发生的微小差异的相关性取决于预期的影响大小和研究问题。