Azqueta Amaya, Muruzabal Damian, Boutet-Robinet Elisa, Milic Mirta, Dusinska Maria, Brunborg Gunnar, Møller Peter, Collins Andrew R
Department of Pharmacology and Toxicology, University of Navarra, C/Irunlarrea 1, 31009 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, Spain.
Department of Pharmacology and Toxicology, University of Navarra, C/Irunlarrea 1, 31009 Pamplona, Spain.
Mutat Res Genet Toxicol Environ Mutagen. 2019 Jul;843:24-32. doi: 10.1016/j.mrgentox.2019.04.007. Epub 2019 May 1.
The comet assay (single cell gel electrophoresis) is widely used as a biomonitoring tool to assess DNA damage - strand breaks, as well as oxidised bases; it can also be adapted to measure DNA repair. It is based on the ability of breaks in the DNA to relax supercoiling, allowing DNA loops to extend from the nuclear core (nucleoid) under an electric field to form a comet-like tail. Most commonly, it is applied to white blood cells. The range of detection is between a few hundred breaks per cell and a few thousand, encompassing levels of damage that can be repaired and tolerated by human cells. Its applications include monitoring various diseases, studying the influence of nutrition on DNA stability, and investigating effects of environmental and occupational mutagens. Here we address the issue of inter-laboratory variation in comet assay results. This variation is largely due to differences in methods. Imposing a standard protocol is not practical, but users should be aware of the crucial parameters that affect performance of the assay. These include the concentration of agarose in which the cells are embedded; the duration of cell lysis, and of enzyme incubation when oxidised bases are being measured; the duration of alkaline unwinding; the duration of electrophoresis and the voltage gradient applied; and the method used to score the comets. Including reference standards in each experiment allows experimental variability to be monitored - and if variation is not extreme, results can be normalised using reference standard values. Reference standards are also essential for inter-laboratory comparison. Finally, we offer recommendations which, we believe, will limit variability and increase the usefulness of this assay in molecular epidemiology.
彗星试验（单细胞凝胶电泳）作为一种生物监测工具被广泛应用于评估DNA损伤——链断裂以及氧化碱基；它也可用于测量DNA修复。该试验基于DNA断裂能够缓解超螺旋的能力，在电场作用下，使得DNA环从核核心（类核）延伸出来，形成彗星状尾巴。最常见的是将其应用于白细胞。检测范围是每个细胞几百个到几千个断裂，涵盖了人类细胞能够修复和耐受的损伤水平。其应用包括监测各种疾病、研究营养对DNA稳定性的影响以及调查环境和职业诱变剂的作用。在此，我们探讨彗星试验结果在不同实验室间存在差异的问题。这种差异很大程度上是由于方法不同所致。实施标准方案并不实际，但使用者应了解影响该试验性能的关键参数。这些参数包括包埋细胞的琼脂糖浓度；细胞裂解的持续时间以及在测量氧化碱基时酶孵育的持续时间；碱性解旋的持续时间；电泳的持续时间和施加的电压梯度；以及用于对彗星进行评分的方法。在每个实验中纳入参考标准可监测实验变异性——如果变异性不太极端，可使用参考标准值对结果进行标准化。参考标准对于实验室间比较也至关重要。最后，我们提出一些建议，我们认为这些建议将限制变异性并提高该试验在分子流行病学中的实用性。
