Schröder Charlotte, Steimer Werner
Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universität München, München, Germany.
PLoS One. 2018 Feb 7;13(2):e0192414. doi: 10.1371/journal.pone.0192414. eCollection 2018.
Epigenetics is believed to provide great chances for a better understanding of the development and treatment of many diseases where the analysis of genomic DNA has so far failed to provide conclusive answers. Methylcytosine is a frequently used quantitative marker of epigenetic studies. Since immediate analysis of sampled material is in most cases not possible, storage time and conditions are critical aspects regarding the quality of genomic DNA and reliability of analysis. Blood is frequently used for such analyses. We, therefore, collected blood samples of ten volunteers and stored them under various conditions for ten months: -70°C, -20°C, 2-8°C and room temperature. An additional aliquot was frozen at -70°C and thawed once a week at room temperature. We then compared the DNA extraction yields and methylation status in relation to storage time and conditions. We found significantly lower DNA extraction yields (up to -97.45%; p ≤ 0.001) as well as significantly higher methylation levels after ten months of storage (up to +42.0%; p ≤ 0.001). These results suggest that storage time has an important influence on DNA analyses of blood samples for all storage conditions. This might be due to differences in stability of methylated and non-methylated DNA. Our study indicates that storage conditions and time may be a critical factor for epigenetic methylation studies and require rigorous validation. For reliable analyses we, therefore, recommend to perform epigenetic analysis directly after sample collection.
表观遗传学被认为为更好地理解许多疾病的发展和治疗提供了巨大机会，而迄今为止，对基因组DNA的分析未能提供确凿答案。甲基胞嘧啶是表观遗传学研究中常用的定量标记。由于在大多数情况下无法对采样材料进行即时分析，储存时间和条件是影响基因组DNA质量和分析可靠性的关键因素。血液经常用于此类分析。因此，我们收集了十名志愿者的血样，并在不同条件下储存了十个月：-70°C、-20°C、2-8°C和室温。另外一份等分试样在-70°C下冷冻，每周在室温下解冻一次。然后，我们比较了与储存时间和条件相关的DNA提取产量和甲基化状态。我们发现，储存十个月后，DNA提取产量显著降低（高达-97.45%；p≤0.001），甲基化水平显著升高（高达+42.0%；p≤0.001）。这些结果表明，对于所有储存条件，储存时间对血样的DNA分析都有重要影响。这可能是由于甲基化和非甲基化DNA稳定性的差异。我们的研究表明，储存条件和时间可能是表观遗传甲基化研究的关键因素，需要严格验证。因此，为了进行可靠的分析，我们建议在样本采集后直接进行表观遗传分析。
