An improved procedure for isolation of high-quality RNA from nematode-infected Arabidopsis roots through laser capture microdissection
An improved procedure for isolation of high-quality RNA from nematode-infected Arabidopsis roots through laser capture microdissection
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DOI：
10.1186/s13007-016-0123-9
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发表时间：
2016-04-26
期刊：
影响因子：
5.1
通讯作者：
Grundler, Florian M. W.
中科院分区：
文献类型：
--
作者：
Anjam, Muhammad Shahzad;Ludwig, Yvonne;Grundler, Florian M. W.
背景资料：胞囊线虫是在寄主植物根部形成专门的摄食结构的活体营养生物，其由肥大细胞的合胞融合体组成。合胞体的形成伴随着感染组织中深刻的转录变化和活跃的代谢。合胞体基因表达研究中的挑战一直是分离纯合胞体材料和随后提取完整RNA。含有合胞体的根片段已用于微阵列分析。然而，包含相邻细胞稀释合胞体特异性mRNA群体。显微切片与激光捕获显微切割（LCM）相结合，为从异质细胞群中分离出摄食位点提供了机会。但是，从合胞体中回收完整的RNA是复杂的，由于延长的步骤固定，组织制备，包埋和sectioning.Results：在本研究中，我们已经优化了LCM的样品制备程序，以分离高质量的RNA从孢囊线虫诱导的拟南芥根合胞体，可用于转录组学研究。我们研究了不同蔗糖浓度作为冷冻保护剂对合胞体切片的RNA质量和形态的影响。我们还比较了各种类型的显微镜载玻片的强粘附性的部分，同时删除包埋material.Conclusion：使用最佳的蔗糖浓度作为冷冻保护起着关键作用，RNA的稳定性和形态的部分。用更高的蔗糖浓度处理可以最大限度地降低RNA降解的风险，而更长的孵育时间有助于保持组织切片的形态。我们的方法允许从线虫摄食位点分离高质量的RNA，适合于下游应用，如微阵列实验。
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.