Abstract

Exosomal Biomarkers in Clinical Diagnostics – How to Identify Valid and Better Biomarker Signatures from Micro-vesicular Small RNA Sequencing

Michael Pfaffl, Professor, Technical University of Munich

Extracellular vesicles (EVs) are circulating in body liquids and are involve in the intercellular communication with key functions in physiological or pathological processes. In recent time especially the exosomes have gained huge interest because of their molecular diagnostic potential based on the containing microRNAs. The past decade has brought about the development and commercialization of a multitude of extraction methods to isolate EVs and exosomes, primarily from blood compartments. The exosome purity and which subpopulations of EVs are captured strongly depend on the applied isolation method, which in turn determines how suitable resulting samples are for potential downstream applications and biomarker discovery. Herein we compared the performance of various optimized isolation principles for serum EVs/exosomes in healthy individuals and critically ill patients. The isolation methods were benchmarked regarding their suitability for biomarker discovery as well as biological characteristics of captured vesicles. Isolated vesicles were deeply characterized by NTA (amount, size, distribution), surface marker proteins (Western Blot), and containing small-RNA families (small-RNA NGS). To analyze the sequencing results, a self-established bioinformatics pipeline for microRNA (based on R) and a deeper analysis of their isoforms (isomiRs) was applied. Goal was the development of microRNA/isomiR biomarker signature for the early diagnosis and for a valid classification of critical ill patients, with focus on sepsis. The results provides guidance for navigating the multitude of EV/exosome isolation methods available, and helps researchers and clinicians in the field of molecular diagnostics to make the right choice about the EV/exosome isolation strategy.