A comet assay protocol for analysis of DNA damage in cryopreserved nuclei from Arabidopsis seeds, seedlings, and leaves.

IF 4.1 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Plant Science Pub Date : 2025-10-09 DOI:10.1016/j.plantsci.2025.112798

José Roberto Torres, Ignacio Lescano López, María Elena Alvarez

{"title":"A comet assay protocol for analysis of DNA damage in cryopreserved nuclei from Arabidopsis seeds, seedlings, and leaves.","authors":"José Roberto Torres, Ignacio Lescano López, María Elena Alvarez","doi":"10.1016/j.plantsci.2025.112798","DOIUrl":null,"url":null,"abstract":"<p><p>The comet assay is classically applied to study DNA damage at the single-cell level. In plants, it is commonly used for the analysis of seedlings, leaves, and seeds. The assay is time-consuming and runs continuously, making it difficult to process large volumes of samples or samples derived from kinetic studies in parallel. To overcome these limitations, we present an optimized protocol of neutral comet assay for analysis of Arabidopsis seeds, seedlings and leaves. It includes the cryopreservation of nuclei under conditions that do not affect DNA integrity. This option is not present in other available protocols for plants and allows samples collected at different times to be preserved and then processed simultaneously. We provide details on all experimental steps, including slide preparation, to obtain high-quality samples without the need of commercial kits. Moreover, we demonstrated its effectiveness in quantifying DNA damage induced by abiotic and biotic stresses. This versatile approach will help to optimize sample processing and will contribute to improving the studies of DNA damage in plants.</p>","PeriodicalId":20273,"journal":{"name":"Plant Science","volume":" ","pages":"112798"},"PeriodicalIF":4.1000,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Science","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/j.plantsci.2025.112798","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

The comet assay is classically applied to study DNA damage at the single-cell level. In plants, it is commonly used for the analysis of seedlings, leaves, and seeds. The assay is time-consuming and runs continuously, making it difficult to process large volumes of samples or samples derived from kinetic studies in parallel. To overcome these limitations, we present an optimized protocol of neutral comet assay for analysis of Arabidopsis seeds, seedlings and leaves. It includes the cryopreservation of nuclei under conditions that do not affect DNA integrity. This option is not present in other available protocols for plants and allows samples collected at different times to be preserved and then processed simultaneously. We provide details on all experimental steps, including slide preparation, to obtain high-quality samples without the need of commercial kits. Moreover, we demonstrated its effectiveness in quantifying DNA damage induced by abiotic and biotic stresses. This versatile approach will help to optimize sample processing and will contribute to improving the studies of DNA damage in plants.

查看原文本刊更多论文

用于分析拟南芥种子、幼苗和叶片中低温保存细胞核DNA损伤的彗星测定方案。

彗星试验通常用于研究单细胞水平的DNA损伤。在植物中，它通常用于分析幼苗、叶子和种子。该分析耗时且连续运行，使得难以处理大量样品或平行动力学研究衍生的样品。为了克服这些限制，我们提出了一种优化的中性彗星测定方案，用于分析拟南芥种子、幼苗和叶片。它包括在不影响DNA完整性的条件下对细胞核进行低温保存。这一选项不存在于其他可用的植物方案中，并允许在不同时间收集的样品保存，然后同时处理。我们提供所有实验步骤的详细信息，包括玻片制备，以获得高质量的样品，而不需要商业试剂盒。此外，我们证明了它在量化非生物和生物胁迫诱导的DNA损伤方面的有效性。这种多用途的方法将有助于优化样品处理，并将有助于改善植物DNA损伤的研究。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Plant Science 生物-生化与分子生物学

CiteScore

9.10

自引率

1.90%

发文量

322

审稿时长

33 days

期刊介绍： Plant Science will publish in the minimum of time, research manuscripts as well as commissioned reviews and commentaries recommended by its referees in all areas of experimental plant biology with emphasis in the broad areas of genomics, proteomics, biochemistry (including enzymology), physiology, cell biology, development, genetics, functional plant breeding, systems biology and the interaction of plants with the environment. Manuscripts for full consideration should be written concisely and essentially as a final report. The main criterion for publication is that the manuscript must contain original and significant insights that lead to a better understanding of fundamental plant biology. Papers centering on plant cell culture should be of interest to a wide audience and methods employed result in a substantial improvement over existing established techniques and approaches. Methods papers are welcome only when the technique(s) described is novel or provides a major advancement of established protocols.