An improved procedure for isolation of high-quality RNA from nematode-infected Arabidopsis roots through laser capture microdissection

Abstract

Background: Cyst nematodes are biotrophs that form specialized feeding structures in the roots of host plants, which consist of a syncytial fusion of hypertrophied cells. The formation of syncytium is accompanied by profound transcriptional changes and active metabolism in infected tissues. The challenge in gene expression studies for syncytium has always been the isolation of pure syncytial material and subsequent extraction of intact RNA. Root fragments containing syncytium had been used for microarray analyses. However, the inclusion of neighbouring cells dilutes the syncytium-specific mRNA population. Micro-sectioning coupled with laser capture microdissection (LCM) offers an opportunity for the isolation of feeding sites from heterogeneous cell populations. But recovery of intact RNA from syncytium dissected by LCM is complicated due to extended steps of fixation, tissue preparation, embedding and sectioning.

Results: In the present study, we have optimized the procedure of sample preparation for LCM to isolate high quality of RNA from cyst nematode induced syncytia in Arabidopsis roots which can be used for transcriptomic studies. We investigated the effect of various sucrose concentrations as cryoprotectant on RNA quality and morphology of syncytial sections. We also compared various types of microscopic slides for strong adherence of sections while removing embedding material.

Conclusion: The use of optimal sucrose concentrations as cryoprotection plays a key role in RNA stability and morphology of sections. Treatment with higher sucrose concentrations minimizes the risk of RNA degradation, whereas longer incubation times help maintaining the morphology of tissue sections. Our method allows isolating high-quality RNA from nematode feeding sites that is suitable for downstream applications such as microarray experiments.

Keywords: Arabidopsis; Cyst nematode; LCM; Nematode; RNA degradation; Root; Syncytium.

Fig. 1

Influence of tissue fixation and embedding on RNA quality. Electropherograms indicating the quality of RNA extracted from Arabidopsis root segments infected with nematodes at 5 dpi. a RNA was extracted from tissues following all steps of fixation, cryoprotection and embedding, however before LCM. Number of root pieces (containing syncytium) used for extraction: 10; RNA concentration: 3407 pg/μL. b Tissue samples after fixation and cryoprotection but before embedding. There is a splice between Ladder and RNA samples; however, both runs are from the same chip and were put together to facilitate visualization. Number of root pieces (containing syncytium) used for extraction: 15; RNA concentration: 4693 pg/μL. c Control samples with fixation but without cryoprotection or embedding. Number of root pieces (containing syncytium) used for extraction: approx. 25 RNA concentration: 1440 pg/μL. a The experiment was performed once. b, c The experiments were repeated three times and data from one representative experiment is provided. S seconds, bp base pair

Fig. 2

Quality assessment of RNA isolation from samples after modifying the tissue preparation steps. a RNA isolated after direct incubation in 15 % sucrose solution (low sucrose concentration treated samples). Number of root pieces (containing syncytium) used for extraction: 10; RNA Concentration: 2514 pg/μL. b RNA isolated after direct incubation in 34 % sucrose (high sucrose concentration treated samples). Number of root pieces (containing syncytium) used for extraction: 10; RNA concentration: 2253 pg/μL. c RNA isolation from samples B after LCM. Number of syncytium used for cryosectioning: 30; RNA concentration: 6297 pg/μL. a–c The experiments were repeated three times and data from one representative experiment is provided. S seconds, bp base pair

Fig. 3

Morphology of longitudinal syncytial samples (10 µm thin)upon different sucrose treatment. a Infected root segments upon direct incubation in 15 % sucrose solution (low sucrose concentration treated samples). b Infected root segments upon direct incubation in 34 % sucrose (high sucrose concentration treated samples). c Infected root segments upon direct overnight incubation in 34 % sucrose. Asterisk (*) nematode, S syncytium, scale bar 100 µM

Fig. 4

Amplification of cDNA, where a–c represents three replications of syncytial samples processed after LCM. The virtual gel generated by an Agilent 2100 Bioanalyzer is shown in d. nt nucelotide, bp base pair

Fig. 5

Analysis of gene expression in uninfected leaf tissues and in 5-days-old syncytium by RT-PCR. Negative control is without template