Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation

Abstract

AbstractTo 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 MicrodissectionBasic Protocol 2: Tissue SectioningBasic Protocol 3: Laser Capture Microdissection of Embedded TissueBasic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples