The genetic profiling of patient material has yielded reliable prognostic biomarkers that are paramount for early detection, diagnosis and assessment of effective therapy. One drawback is that patient tissue is not always available or accessible without conducting timely and painful procedures. The presence of small genetic elements called ‘microRNAs’ in extracellular vesicles (EV) or stably bound to proteins in patient biofluids opens unique possibilities for using such circulating RNAs as easily accessible genetic biomarkers. Our laboratory uses multiple strategies for development of ‘liquid biopsy’ tests that should enable non-invasive diagnosis of cancer in the future, for instance by a simple blood test. We are using similar strategies in severe autoimmune patients with chronic inflammation and discovered a link between EV physiology (exosomes), Epstein Barr virus infection and antiviral immunity. For these studies we use the latest high-throughput next-generation sequencing techniques to unravel the complete small RNA landscape of exosomes. We have found fundamental differences between small RNAs in tissues/cell when compared to exosomes, pointing to a function outside the cell in which they were produced. Currently we investigate the possibility that small distinctions in extracellular small RNA content maybe applicable for non-invasive disease monitoring, primarily by isolation of exosomes from patient blood and urine (van Eijndhoven et al., 2016).
Development of novel Next Generation Sequencing methods Secreted microRNAs (miRNAs) are proposed as powerful disease indicators and when associated with extracellular vesicles (EVs), have an active role in organ crosstalk. Because the majority of miRNAs are broadly expressed across cell-types and tissues, a single miRNA is rarely informative for a disease condition. Recent advances show that non-templated nucleotide additions (isomiRs) change miRNA gene-regulatory functions and incorporating this additional layer of complexity increases diagnostic accuracy of miRNAs. Small RNA sequencing is the only method that gains comprehensive insight into the diversity and relative quantities of miRNAs and their subspecies. We show here that, despite improvements, current small RNAseq methods remain imprecise. The accuracy of cell-free miRNA detection is particularly low due to low input amounts which may underlie difficulties in demonstrating clinical utility. The method we developed (isoSeq) makes use of 5N-randomized 3’ and 5’ adapters and unique molecular identifier (UMI) dramatically reducing bias in detection. With custom-designed synthetic isomiR spike-ins and cell-lines lacking terminal uridylases (TUT4 and TUT7), we validated that isoSeq provides robust, quantitative and qualitative information on miRNA complexity from circulating EVs, an increasingly used liquid biosource. IsoSeq will improve our understanding of miRNA biology and their adaption as minimally-invasive disease indicators.