Sequencing of miRNAs isolated from exosomes has great potential to identify novel disease biomarkers, but exosomes have low amount of RNA, hindering adequate analysis and quantification. Here, we have assessed several steps in developing an optimized small RNA (sRNA) library preparation protocol for next-generation sequencing (NGS) miRNA analysis from urinary exosomes. A total of 24 urinary exosome samples from donors were included in this study. RNA was extracted by column-based methods. The quality of extracted RNA was assessed by spectrophotometric quantification and Bioanalyzer software analysis. All libraries were prepared using the CleanTag small RNA library preparation protocol and the effect of our additional modifications on adapter-dimer presence, sequencing data and tagged small RNA library population was also analyzed. Our results show that good quality sequencing libraries can be prepared following our optimized small RNA library preparation protocol from urinary exosomes. When the size selection by gel purification step was included within the workflow, adapter-dimer was totally removed from cDNA libraries. Furthermore, the inclusion of this modification step within small RNA library protocol augmented the small RNA mapped reads, with an especially significant 37% increase in miRNA reads, and the gel purification step made no difference to the tagged miRNA population. This study provides researchers with an optimized small RNA library preparation workflow for next generation sequencing based exosome-associated miRNA analysis that yields a high amount of miRNA mapped reads without skewing the tagged miRNA population significantly.

中文翻译：

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优化人尿液外泌体的小RNA文库制备方案。

从外泌体分离的miRNA的测序具有识别新型疾病生物标记物的巨大潜力，但外泌体的RNA含量低，从而妨碍了适当的分析和定量。在这里，我们评估了开发优化的小RNA（sRNA）文库制备方案的几个步骤，用于从尿液外泌体进行下一代测序（NGS）miRNA分析。这项研究总共包括了来自供体的24个尿液外泌体样品。通过基于柱的方法提取RNA。通过分光光度定量和Bioanalyzer软件分析评估提取的RNA的质量。所有文库均使用CleanTag小RNA文库制备方案制备，还分析了我们的其他修饰对衔接子二聚体存在，测序数据和标记的小RNA文库种群的影响。我们的结果表明，可以按照我们优化的尿液外泌体小RNA文库制备方案制备高质量的测序文库。当通过凝胶纯化步骤进行大小选择包括在工作流程中时，衔接子二聚体已从cDNA文库中完全去除。此外，在小RNA文库方案中包含此修饰步骤可增加小RNA定位图的读取，尤其是miRNA读数显着增加37％，并且凝胶纯化步骤对标记的miRNA群体没有影响。这项研究为研究人员提供了优化的小RNA库制备工作流程，可用于基于下一代测序的与外来体相关的miRNA分析，该分析可产生大量的miRNA映射读段，而不会显着倾斜标记的miRNA群体。