Abstract
BackgroundSequencing 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.MethodsA 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.ResultsOur 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.ConclusionsThis 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.
Highlights
Sequencing of miRNAs isolated from exosomes has great potential to identify novel disease biomark‐ ers, but exosomes have low amount of RNA, hindering adequate analysis and quantification
Modification of small RNA protocol does not suppress adapter‐dimer formation The small RNA library prep kit allowed us to obtain the corresponding libraries for next-generation sequencing from urinary exosomes
Electropherograms obtained for DNA Fragment Analysis by Capillary Electrophoresis showed a peak at 110–120 bp that corresponds to the amount of adapter-dimer and other peaks at 135–155 bp that correspond to small RNA types, miRNA and other sRNA
Summary
Sequencing of miRNAs isolated from exosomes has great potential to identify novel disease biomark‐ ers, but exosomes have low amount of RNA, hindering adequate analysis and quantification. Over recent years changes in circulating microRNAs (miRNAs) levels have been associated with a broad range of pathological processes. Their analysis offers various advantages which make them a potential goldmine in identification of novel biomarkers: (1) they can be found in non- or minimally invasive specimens; (2) they are relatively stable in clinical samples as regards RNase digestion, temperature variation and multiple freeze– thaw cycles; (3) they are involved in pathway regulation, Olivares et al J Transl Med (2020) 18:132 showing tissue and cell-specific expression profiles [1]. There is growing interest in their use as non-invasive biomarkers for disease diagnosis and for monitoring disease recurrence, overall comprehensive analysis of the entire miRNA repertoire of exosomes in important diseases such as cancer, immune disorders and cardiovascular disease [14,15,16]
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