Olivares Dolores, Perez-Hernandez Javier, Perez-Gil Daniel, Chaves Felipe J, Redon Josep, Cortes Raquel
Cardiometabolic and Renal Risk Research Group, INCLIVA Biomedical Research Institute, Avd. Menéndez Pelayo, accesorio 4, 46010, Valencia, Spain.
INSERM, U1016, Cochin Institute, 75014, Paris, France.
J Transl Med. 2020 Mar 18;18(1):132. doi: 10.1186/s12967-020-02298-9.
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.
对从外泌体中分离出的微小RNA（miRNA）进行测序，在识别新型疾病生物标志物方面具有巨大潜力，但外泌体中的RNA含量较低，这阻碍了充分的分析和定量。在此，我们评估了开发一种优化的小RNA（sRNA）文库制备方案的几个步骤，该方案用于对尿外泌体进行下一代测序（NGS）miRNA分析。
本研究共纳入了来自供体的24份尿外泌体样本。采用基于柱的方法提取RNA。通过分光光度法定量和生物分析仪软件分析评估提取RNA的质量。所有文库均使用CleanTag小RNA文库制备方案制备，并且还分析了我们额外修改对衔接子二聚体存在、测序数据和带标签的小RNA文库群体的影响。
我们的结果表明，按照我们优化的小RNA文库制备方案，可以从尿外泌体制备出高质量的测序文库。当在工作流程中纳入凝胶纯化步骤进行大小选择时，衔接子二聚体从cDNA文库中被完全去除。此外，在小RNA文库方案中纳入此修改步骤增加了小RNA比对读数，其中miRNA读数尤其显著增加了37%，并且凝胶纯化步骤对带标签的miRNA群体没有影响。
本研究为研究人员提供了一种优化的小RNA文库制备工作流程，用于基于下一代测序的外泌体相关miRNA分析，该流程可产生大量miRNA比对读数，且不会显著扭曲带标签的miRNA群体。
