MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology

Abstract

Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathologicalprocesses and fundamental biological pathways and responses. Their presence inbiofluids makes them particularly attractive for biomarker identification. However,a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNAprofiles of n = 64 EV low concentration RNA samples (180-49125 pg), isolatedfrom serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based onthree levels of limit of detection stringency, and differential microRNA expressionanalysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can besuccessfully profiled using nCounter microRNA assays, demonstrating acceptableoutput ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels ofinput RNA, high-level differential expression analysis between biological subgroupsidentified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profilingof low abundance EV-derived microRNA, and may provide a solution for researchstudies that focus on limited sample material.