Abstract

Development of a Sensitive and Robust NGS Protocol for microRNA Sequencing in Exosome and Extracellular Vesicle Samples

Laura Klitten, Global Product Manager, Exiqon A/S

The contents of exosomes are subject of investigative efforts worldwide, from basic research to biomarker discovery, with the goal of using insights for diagnostic and therapeutic purposes. The profile of microRNAs contained in exosomes can elucidate cellular functions and responses in diseased and healthy states. Plasma-derived exosomes are commonly isolated by differential centrifugation, which has proven to be difficult and is susceptible to variability in experience and skill of the person performing the isolation. To assess and optimize methodologies for the isolation and characterization of microRNAs in liquid biopsies, plasma samples from three immunized animals were prepared for next generation sequencing using three different methods: Exosome preparation by differential centrifugation, exosome preparation with the miRCURY™ isolation technology, and no exosome prep -RNA was isolated directly from the plasma sample. Library preparation and subsequent sequencing was successful for all preparation methods, with mapping percentages above 75% for all samples. Overall, provided that protocols optimized for liquid biopsies are used, there is good agreement between the microRNA expression patterns detected from whole plasma and isolated exosomes. This consistency across preparation methods attests to the high-quality and robust results that Exiqon Services achieves through experience and implementation of carefully designed and standardized protocols.