Pellicer Jaume, Leitch Ilia J
Jodrell Laboratory, Royal Botanic Gardens, Surrey, UK.
Methods Mol Biol. 2014;1115:279-307. doi: 10.1007/978-1-62703-767-9_14.
Over the years, the amount of DNA in a nucleus (genome size) has been estimated using a variety of methods, but increasingly, flow cytometry (FCM) has become the method of choice. The popularity of this technique lies in the ease of sample preparation and in the large number of particles (i.e., nuclei) that can be analyzed in a very short period of time. This chapter presents a step-by-step guide to estimating the nuclear DNA content of plant nuclei using FCM. Attempting to serve as a tool for daily laboratory practice, we list, in detail, the equipment required, specific reagents, and buffers needed, as well as the most frequently used protocols to carry out nuclei isolation. In addition, solutions to the most common problems that users may encounter when working with plant material and troubleshooting advice are provided. Finally, information about the correct terminology to use and the importance of obtaining chromosome counts to avoid cytological misinterpretations of the FCM data are discussed.
多年来，人们使用多种方法来估计细胞核中的DNA含量（基因组大小），但流式细胞术（FCM）越来越成为首选方法。这项技术的受欢迎之处在于样品制备简便，且能在极短时间内分析大量颗粒（即细胞核）。本章介绍了使用流式细胞术估计植物细胞核DNA含量的详细步骤指南。为了作为日常实验室操作的工具，我们详细列出了所需设备、特定试剂和缓冲液，以及进行细胞核分离最常用的方案。此外，还提供了用户在处理植物材料时可能遇到的最常见问题的解决方案及故障排除建议。最后，讨论了正确术语的使用信息以及获得染色体计数以避免对流式细胞术数据进行细胞学错误解读的重要性。
