Chavan Suchitra, Schnabel Elise, Saski Christopher, Frugoli Julia
Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina.
Department of Plant and Environmental Sciences, Clemson University, Clemson, South Carolina.
Curr Protoc. 2023 Jul;3(7):e844. doi: 10.1002/cpz1.844.
To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique requires neither separation of cells by enzymatic digestion of any kind nor cell-specific reporter genes, and it allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capturing of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue Preparation for Laser Capture Microdissection Basic Protocol 2: Tissue Sectioning Basic Protocol 3: Laser Capture Microdissection of Embedded Tissue Basic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples.
为了研究特定时间点单个植物细胞的转录组，我们开发了植物组织固定、包埋和切片的方案，随后进行激光捕获显微切割（LCM）并进行RNA回收处理。LCM可从异质组织切片中分离出单个细胞类型，特别适用于植物处理，因为它不需要破坏细胞壁。这种方法能够准确分离少量细胞，可用于研究不同组织或细胞层中的基因表达谱。该技术既不需要通过任何类型的酶消化来分离细胞，也不需要细胞特异性报告基因，并且允许在捕获前将固定和包埋的组织保存数月。我们所描述的植物细胞固定、包埋、切片和捕获方法可产生高质量的RNA，适用于制备RNA测序文库。© 2023作者。由Wiley Periodicals LLC出版的《当前方案》。基本方案1：激光捕获显微切割的组织制备 基本方案2：组织切片 基本方案3：包埋组织的激光捕获显微切割 基本方案4：从激光捕获显微切割样品中提取RNA。
