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

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.