Blood DNA Yield but Not Integrity or Methylation Is Impacted After Long-Term Storage
Blood DNA Yield but Not Integrity or Methylation Is Impacted After Long-Term Storage
复制标题
DOI：
10.1089/bio.2015.0045
复制
发表时间：
2016-02-01
影响因子：
1.6
通讯作者：
Lescuyer, Pierre
中科院分区：
文献类型：
--
作者：
Bulla, Alexandre;De Witt, Brian;Lescuyer, Pierre
收集人类全血提取基因组DNA是众多临床研究的一部分。由于DNA提取并不总是在样本采集时进行，因此全血样本在处理前可能要储存数年。因此，使用适当的储存条件对于获得足够数量和足够质量的DNA至关重要，以便从随后的分子生物学分析中获得可靠的结果。本研究采集了8名健康志愿者的EDTA全血样本，比较了不同的保存时间（最长1年）和保存温度（室温、4℃、-20℃、-80℃）。在储存前后，还对添加DNA防腐剂的效果进行了评估。用紫外分光光度法和荧光光谱法测定DNA浓度，计算DNA提取率和双链DNA比。DNA完整性由琼脂糖凝胶电泳和远程聚合酶链反应控制。同时还评估了储存条件对DNA甲基化的影响。结果表明，一定的储存条件对DNA提取率有显著影响，但对DNA完整性和甲基化的影响很小或没有影响。EDTA血液在-80℃下的储存保证了高质量的DNA和良好的产量。在解冻储存在-20℃或-80℃的EDTA血液之前，加入DNA防腐剂可以获得更高的DNA提取率。在DNA防腐剂存在的情况下，在室温下长期储存似乎也是一种可靠的方法。
Collection of human whole blood for genomic DNA extraction is part of numerous clinical studies. Since DNA extraction cannot always be performed at the time of sample collection, whole blood samples may be stored for years before being processed. The use of appropriate storage conditions is then critical to obtain DNA in sufficient quantity and of adequate quality in order to obtain reliable results from the subsequent molecular biological analyses. In this study, EDTA whole blood samples were collected from 8 healthy volunteers, and different durations (up to 1 year) and temperatures (room temperature, 4 degrees C, -20 degrees C, and -80 degrees C) of storage were compared. The effect of the addition of a DNA preservative agent was also assessed before and after storage. DNA concentrations measured by UV spectrophotometry and spectrofluorometry were used to calculate DNA extraction yields and double-strand DNA ratios. DNA integrity was controlled by agarose gel electrophoresis and long-range polymerase chain reaction. The impact of storage conditions on DNA methylation was also evaluated. Results showed that certain storage conditions have a significant impact on the DNA extraction yield but little or no effect on DNA integrity and methylation. Storage of EDTA blood at -80 degrees C guarantees high-quality DNA with a good yield. Higher DNA extraction yields were obtained with the addition of a DNA preservative agent before thawing EDTA blood stored at -20 degrees C or -80 degrees C. Long-term storage at room temperature in the presence of a DNA preservative agent also appeared to be a reliable procedure.