Laser Capture Micro-Dissection Coupled to RNA Sequencing: A Powerful Approach Applied to the Model Legume Medicago truncatula in Interaction with Sinorhizobium meliloti

Abstract

Understanding the development of multicellular organisms requires the identification of regulators, notably transcription factors, and specific transcript populations associated with tissue differentiation. Laser capture microdissection (LCM) is one of the techniques that enable the analysis of distinct tissues or cells within an organ. Coupling this technique with RNA sequencing (RNAseq) makes it extremely powerful to obtain a genome-wide and dynamic view of gene expression. Moreover, RNA sequencing allows two or potentially more interacting organisms to be analyzed simultaneously. In this chapter, a LCM-RNAseq protocol optimized for root and symbiotic root nodule analysis is presented, using the model legume Medicago truncatula (in interaction with Sinorhizobium meliloti in the nodule samples). This includes the description of procedures for plant material fixation, embedding, and micro-dissection; it is followed by a presentation of techniques for RNA extraction and amplification, adapted for the simultaneous analysis of plant and bacterial cells in interaction or, more generally, polyadenylated and non-polyadenylated RNAs. Finally, step-by-step statistical analyses of RNAseq data are described. Those are critical for quality assessment of the whole procedure and for the identification of differentially expressed genes.

An optimized protocol for isolation of high‐quality RNA through laser capture microdissection of leaf material

“…Thus, paradermal sectioning resulted in more tissue being captured per slide (Figure a,c), was about twice as quick, and so reduced the risk of RNA degradation. However, consistent with reports on other tissues (Roux et al., ), we also found that RNA quality from paraffin‐embedded tissue was low. Since the fixation process itself appeared not to have a deleterious effect on RNA quality (Figure ), we reasoned that losses in RNA integrity were caused by fragmentation occurring at the relatively high temperatures associated with infiltration of paraffin wax.…”

“…Typically, in these studies, non‐cross‐linking solutions such as Farmer's fixative or acetone are used to stabilize RNA, and the dehydrated tissue is then mounted in paraffin wax at ~60°C to allow thin sections to be subjected to microdissection. Although histological details are well preserved using this method, considerable RNA degradation can take place (Gomez et al., ; Roux, Rodde, Moreau, Jardinaud, & Gamas, ). We found this to be a particular problem with low abundance cell types of leaves.…”

An optimised protocol for isolation of RNA through laser capture microdissection of leaf material

“…Thus, paradermal sectioning resulted in more tissue being captured per slide 153 ( Figure 1A and 1C), was about twice as quick, and so reduces the risk of RNA degradation. However, 154 consistent with reports on other tissues (Roux et al 2018), we also found that RNA quality from 155 paraffin embedded tissue was low. Since the fixation process itself appeared not to have a 156 deleterious effect on RNA quality (Supplemental Figure 1), we reasoned that losses in RNA integrity 157 were caused by fragmentation occurring at the relatively high temperatures associated with 158 infiltration of paraffin wax.…”

“…In the case of leaves, the process of protoplasting 52 is known to generate a significant stress response and de-differentiation (Sawers et al 2007) such 53 that this approach is compromised if the aim is to better understand photosynthesis. In principle, 54 laser capture microdissection provides an orthogonal method to these approaches, enabling highly 55 purified cell populations to be harvested without requiring the generation of transgenic lines (Nelson 56 Although histological details are well preserved using this method, considerable RNA degradation 69 can take place (Gomez et al 2009;Roux et al 2018). We found this to be a particular problem with 70 low abundance cell types of leaves.…”

Transcriptional Reprogramming of Legume Genomes: Perspective and Challenges Associated With Single-Cell and Single Cell-Type Approaches During Nodule Development

“…In indeterminate nodules, Roux et al (2014) collected different zones of the M. truncatula nodules validating the use of laser microdissection in order to better depict the unique transcriptional properties of each zone. This method helps validating the used of laser microdissection to enhance the purity of the biological samples used from nodules ( Roux et al, 2018 ). More recently, the same group revealed the role of Mt DME ( DEMETER ) as a major regulator of the transcriptional activity of nodule genes and transposable elements ( Satge et al, 2016 ).…”