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The poster and abstract below were presented at Biomarkers Series UK 2020 (Manchester, UK)
The human body is comprised of many different types of biofluids, including blood plasma and urine, that are frequently used for liquid biopsy-based diagnosis of a disease or for monitoring patients' response to therapy. We demonstrate that all these biofluids are rich sources of extracellular RNA, such as messenger RNAs, long non-coding RNAs, circular RNAs, and microRNAs. We put forward that RNA has particular advantages as a precision medicine biomarker because of its dynamic abundance reflecting cellular states in health and disease. We have developed and benchmarked two RNA sequencing workflows for extracellular RNA from biofluids, focusing on mRNA or total RNA, respectively. The inclusion of two sets of spike-in RNA controls during RNA extraction or library preparation serve as workflow controls, and also allows accurate assessment of the RNA concentration. We reproducibly detect a few thousand genes in biofluids and derived extracellular vesicles, and see enrichment of circular RNAs compared to tissues. Each biofluid transcriptome is also enriched for RNA molecules derived from specific tissues and cell types, enabling a more informed selection of the most relevant biofluid to study particular diseases. To assess the biomarker potential in biofluids, four cohorts representing a broad spectrum of diseases were profiled, revealing differential gene abundances between case and control subjects. Finally, several pre-analytical variables in the blood plasma RNA sequencing workflow were systematically studied, including RNA extraction kits, blood collection tubes, and varying durations between blood draw and plasma preparation. RNA purification kits show substantial differences in performance in terms of reproducibility, sensitivity, and observed transcriptome complexity. The blood collection tubes display marked differences in biotype composition, whereby tubes with preservation reagents do not stabilize RNA very well, as reflected by increasing RNA concentrations and numbers of detected genes over time. These findings are crucially important to improve and standardize future RNA-based liquid biopsy-guided precision medicine applications.