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Unbiased approach to profile the variety of small non-coding RNA of human blood plasma with massively parallel sequencing technology

机译：使用大规模并行测序技术对人血浆中各种小非编码RNA进行分析的无偏方法

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Objective: Understanding structures of circulating RNA expands fundamental knowledge of cell communications and signaling pathways as well as allows developing new molecular diagnostic approaches. The aim of this study was to deploy a new approach to sequencing cDNA library construction which expands the capabilities of high-throughput sequencing analysis of small non-coding RNAs. With the approach, we performed massively parallel sequencing of human blood plasma RNA to document profile of common and peculiar RNA species normally circulating in blood of healthy individuals. Methods: Total RNA was extracted from blood plasma samples of eight apparently healthy individuals. To obtain comprehensive cDNA libraries RNA was dephosphorylated and then 5'-phosphorylated. 5'-Phosphorylated total plasma RNA was ligated with adapters, reverse transcribed and eight personalized cDNA libraries were constructed. Libraries were sequenced with SOLiD~(TM) technology. Results/conclusion: Fragments of rRNA, mitochondrial transcripts, microRNAs, fragments of scRNAs, snRNA and snoRNA, fragments of several mRNAs as well as the set of newly discovered transcripts were found to be permanent representatives of human blood plasma RNAs. Advanced mapping allowed to identify circulating herpes virus and enterobacterial transcripts. Documented profile of circulating RNA of healthy individuals provides basis for development of new approaches in research and diagnosis of human pathology.
机译：目的：了解循环RNA的结构可扩展细胞通讯和信号传导途径的基础知识，并允许开发新的分子诊断方法。这项研究的目的是采用一种新的方法来测序cDNA文库，从而扩展了对小型非编码RNA的高通量测序分析的能力。通过这种方法，我们对人类血浆RNA进行了大规模并行测序，以记录正常情况下在健康个体血液中循环的常见和特殊RNA种类的概况。方法：从八名看上去健康的个体的血浆样本中提取总RNA。为了获得全面的cDNA文库，将RNA去磷酸化，然后5'-磷酸化。将5'-磷酸化的总血浆RNA与衔接子连接，逆转录并构建了八个个性化cDNA文库。使用SOLiDTM技术对文库进行测序。结果/结论：rRNA片段，线粒体转录物，microRNA，scRNA片段，snRNA和snoRNA片段，几种mRNA片段以及一组新发现的转录本被发现是人类血浆RNA的永久代表。先进的作图可以识别循环疱疹病毒和肠细菌转录本。健康人循环RNA的文献资料为人类病理学研究和诊断新方法的开发提供了基础。

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