Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay
Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay
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DOI：
10.1093/mutage/get049
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发表时间：
2013-11-01
期刊：
影响因子：
2.7
通讯作者：
Wallin, Hakan
中科院分区：
文献类型：
--
作者：
Jackson, Petra;Pedersen, Lourdes M.;Wallin, Hakan
组织和细胞中DNA链断裂水平的彗星分析已成为筛选遗传毒性的常用方法。绝大多数已发表的研究使用了新鲜组织和采集后立即处理的细胞。然而，我们已经使用冷冻组织和细胞超过10年，我们相信冷冻样品可以提高该方法的效率。我们比较了暴露于已知诱变剂甲磺酸甲酯（0，25，75，112.5mg/kg）的小鼠新鲜和冷冻支气管肺泡细胞以及肺和肝组织中的DNA链断裂水平。我们使用了高通量彗星协议与DNA链断裂水平的全自动评分。新鲜和冷冻样本的总体结果一致[尾部DNA %（%TDNA）的R-2为0.93，尾部长度（TL）的R-2为0.78]。在溶剂对照组的肺和肝组织中观察到%TDNA略微增加;在高剂量组的支气管肺泡灌洗液细胞中观察到TL略微减少。在我们的彗星方案中，在组织收集时立即冷冻指定用于彗星分析的一小块组织，并保持深度冷冻，直到快速均质化并包埋在琼脂糖中。为了证明长期冷冻样本的可行性，我们分析了10年内收集的内部历史阴性和阳性彗星试验对照品的日常变化（1128次观察，11批冷冻的未处理和H2 O2处理的A549肺上皮细胞）。H_2O_2处理解释了大部分的变异57.77%，日间变异仅为21.2%。所提出的方案允许分析在较长的时间跨度，在不同的位置，减少变异，减少数量的样品，是适合毒理学和流行病学研究。然而，使用冷冻组织需要在分析前的准备过程中非常小心，处理是一个主要的风险因素。
The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [R-2 0.93 for %DNA in tail (%TDNA) and R-2 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H2O2-treated A549 lung epithelial cells). The H2O2 treatment explained most of the variation 5777% and the day-to-day variation was only 212%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor.