An improved procedure for isolation of high-quality RNA from nematode-infected Arabidopsis roots through laser capture m
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Plant Methods Open Access
METHODOLOGY
An improved procedure for isolation of high‑quality RNA from nematode‑infected Arabidopsis roots through laser capture microdissection Muhammad Shahzad Anjam1,4, Yvonne Ludwig2, Frank Hochholdinger2, Chisato Miyaura3, Masaki Inada3, Shahid Siddique1 and Florian M. W. Grundler1*
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: Syncytium, LCM, Root, Arabidopsis, RNA degradation, Cyst nematode, Nematode Background Plant-parasitic nematodes (PPNs) are obligate biotrophs that cause significant damage to almost every economically important crop [27]. PPNs are classified based on their feeding habits as either ectoparasites or
*Correspondence: grundler@uni‑bonn.de 1 INRES ‑ Molecular Phytomedicine, Rheinische Friedrich-WilhelmsUniversitaet Bonn, Karlrobert‑Kreiten‑Straße 13, 53115 Bonn, Germany Full list of author information is available at the end of the article
endoparasites. The endoparasitic root-knot (Meloidogyne spp.) and cyst nematodes (Globodera spp. and Heterodera spp.) are sedentary parasites of roots and the primary nematode pathogen of a wide range of crops. Infective stage juveniles (J2s) of cyst nematodes invade the host root and migrate intracellularly until they r
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