Casavant N Carol, Kuhl Joseph C, Xiao Fangming, Caplan Allan B, Dandurand Louise-Marie
Department of Plant, Soil, and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339.
J Nematol. 2017 Mar;49(1):12-20. doi: 10.21307/jofnem-2017-041.
The introduction of high-throughput sequencing technologies has made transcriptome analyses of plant-pathogen interactions almost routine. Nevertheless, it is still challenging to obtain RNA from populations made up of two species. An RNA extraction method that worked well on free-living failed when applied to isolated J2 larva. Furthermore, alternative protocols that extracted RNA from free-living J2 larva produced less satisfactory results once the animals entered their hosts' roots. We have compared several extraction procedures to ascertain whether a single protocol was capable of recovering high-quality, high-molecular-weight RNA from newly hatched J2 larva as well as from larva embedded in roots of both potatoes ( L. cv. Desiree) and a very distantly related species, . Although it was possible to recover large amounts of RNA from J2 larvae using Proteinase K treatments, this protocol failed to yield high-quality nematode RNA from infected roots. By comparison, mechanical disruption procedures yielded lower amounts of RNA from infected roots, but what was recovered was of higher quality. We conclude that different extraction protocols need to be developed to sample mixed populations of organisms.
高通量测序技术的引入使得植物与病原体相互作用的转录组分析几乎成为常规操作。然而，从由两个物种组成的群体中获取RNA仍然具有挑战性。一种对自由生活的[生物]有效的RNA提取方法应用于分离出的J2幼虫时却失败了。此外，从自由生活的J2幼虫中提取RNA的替代方案，一旦这些动物进入宿主根部，其结果就不那么令人满意了。我们比较了几种提取程序，以确定是否有一种单一的方案能够从新孵化的J2幼虫以及嵌入马铃薯（L. cv. Desiree）和一个亲缘关系很远的物种[具体物种名缺失]根部的幼虫中回收高质量、高分子量的RNA。尽管使用蛋白酶K处理可以从J2幼虫中回收大量RNA，但该方案未能从受感染的根部产生高质量的线虫RNA。相比之下，机械破碎程序从受感染的根部产生的RNA量较低，但回收的RNA质量较高。我们得出结论，需要开发不同的提取方案来对混合生物群体进行取样。
