Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge, Sweden.
Mutagenesis. 2010 Mar;25(2):125-32. doi: 10.1093/mutage/gep055. Epub 2009 Nov 30.
The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/10(6) bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose-response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.
单细胞凝胶电泳（彗星试验）的应用日益广泛，这突出了其作为检测 DNA 损伤的方法的普及性，包括使用酶来评估氧化损伤的 DNA。然而，由于实验方案（例如裂解条件、酶处理、碱处理时间和电泳）和用于报告结果的终点（例如尾部的 %DNA、任意单位、尾部矩和尾部长度）存在差异，因此不同实验室之间比较 DNA 损伤水平可能较为困难。一种促进比较的方法是通过用电离辐射校准将初级彗星试验终点转换为每 10（6）bp 的损伤数。本研究旨在研究使用 FPG（甲酸嘧啶 DNA 糖基化酶）将氧化嘌呤转化为链断裂来评估彗星试验中氧化损伤 DNA 的实验室间变异性。将用不同浓度的光敏剂（Ro 19-8022）加光处理诱导 DNA 氧化损伤并经电离辐射校准的编码样本分发给 10 个参与实验室，用其各自的彗星试验方案测量 DNA 损伤。10 个实验室中有 9 个报告了编码样本中损伤水平的相同排序。氧化损伤 DNA 的评估差异主要归因于方案的差异。使用实验室特定的校准曲线将数据转换为损伤/10（6）bp 后，实验室间的差异减少了。光敏剂浓度对净 FPG 敏感部位变异的贡献从 49%增加到 73%，而实验室间的变异减少。参与实验室成功地在编码样本中找到了氧化损伤 DNA 的剂量反应，但仍需要标准化方案，以便能够在实验室之间进行直接比较。
