Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq.
Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq.
复制标题
DOI：
10.3791/50093
复制
发表时间：
2013-04
期刊：
影响因子：
--
通讯作者：
Phanidhar Kukutla;Matthew J. Steritz;Jiannong Xu
中科院分区：
文献类型：
--
作者：
Phanidhar Kukutla;Matthew J. Steritz;Jiannong Xu
蚊子的肠道容纳了昆虫生命周期不同阶段的动态微生物群落。肠道群落的遗传能力和功能的表征将为肠道微生物群对蚊子生活特征的影响提供深入了解。宏基因组RNA-Seq已成为分析微生物群落中各种微生物转录组的重要工具。信使RNA通常仅占总RNA的1-3%，而rRNA约占90%。从宏基因组微生物RNA样品中富集信使RNA是具有挑战性的，因为大多数原核mRNA种类缺乏稳定的聚（A）尾。这防止了寡聚d（T）介导的mRNA分离。在这里，我们描述了一种方案，该方案采用样品衍生的rRNA捕获探针从宏基因组总RNA样品中去除rRNA。开始，从宏基因组群落DNA样品扩增蚊子和微生物的小亚基和大亚基rRNA片段。然后，使用T7 RNA聚合酶在体外合成群落特异性生物素化的反义核糖体RNA探针。生物素化rRNA探针与总RNA杂交。通过链霉亲和素包被的珠捕获杂交体并从总RNA中除去。这种基于消减的方案有效地从总RNA样品中去除蚊子和微生物rRNA。将富含mRNA的样品进一步加工用于RNA扩增和RNA-Seq。
The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.