Luna-García Valentín, de Folter Stefan
Unidad de Genómica Avanzada (UGA-Langebio), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav), Irapuato, Mexico.
Bio Protoc. 2024 Sep 5;14(17):e5056. doi: 10.21769/BioProtoc.5056.
For obtaining insights into gene networks during plant reproductive development, having transcriptomes of specific cells from developmental stages as starting points is very useful. During development, there is a balance between cell proliferation and differentiation, and many cell and tissue types are formed. While there is a wealth of transcriptome data available, it is mostly at the organ level and not at specific cell or tissue type level. Therefore, methods to isolate specific cell and tissue types are needed. One method is fluorescent activated cell sorting (FACS), but it has limitations such as requiring marker lines and protoplasting. Recently, single-cell/nuclei isolation methods have been developed; however, a minimum amount of genetic information (marker genes) is needed to annotate/predict the resulting cell clusters in these experiments. Another technique that has been known for some time is laser-assisted microdissection (LAM), where specific cells are microdissected and collected using a laser mounted on a microscope platform. This technique has advantages over the others because no fluorescent marker lines must be made, no marker genes must be known, and no protoplasting must be done. The LAM technique consists in tissue fixation, tissue embedding and sectioning using a microtome, microdissection and collection of the cells of interest on the microscope, and finally RNA extraction, library preparation, and RNA sequencing. In this protocol, we implement the use of normal slides instead of the membrane slides commonly used for LAM. We applied this protocol to obtain the transcriptomes of specific tissues during the development of the gynoecium of . Key features • Laser-assisted microdissection (LAM) allows the isolation of specific cells or tissues. • Normal slides can be used for LAM. • It allows the identification of the transcriptional profiles of specific tissues of the gynoecium.
为了深入了解植物生殖发育过程中的基因网络，以发育阶段特定细胞的转录组作为起点非常有用。在发育过程中，细胞增殖和分化之间存在平衡，会形成许多细胞和组织类型。虽然有大量的转录组数据可用，但大多是器官水平的，而非特定细胞或组织类型水平的。因此，需要分离特定细胞和组织类型的方法。一种方法是荧光激活细胞分选（FACS），但它有局限性，比如需要标记系和原生质体化。最近，单细胞/细胞核分离方法已经得到发展；然而，在这些实验中注释/预测所得细胞簇需要最少数量的遗传信息（标记基因）。另一种已知了一段时间的技术是激光辅助显微切割（LAM），即使用安装在显微镜平台上的激光对特定细胞进行显微切割并收集。这项技术比其他技术有优势，因为无需构建荧光标记系，无需知道标记基因，也无需进行原生质体化。LAM技术包括组织固定、使用切片机进行组织包埋和切片、在显微镜下对感兴趣的细胞进行显微切割和收集，最后进行RNA提取、文库制备和RNA测序。在本方案中，我们采用普通载玻片而非LAM常用的膜载玻片。我们应用此方案来获取 雌蕊发育过程中特定组织的转录组。关键特性 • 激光辅助显微切割（LAM）可分离特定细胞或组织。 • 普通载玻片可用于LAM。 • 它能鉴定 雌蕊特定组织的转录谱。
