Urinary extracellular vesicles: Assessment of pre‐analytical variables and development of a quality control with focus on transcriptomic biomarker research

Abstract

Urinary extracellular vesicles (uEV) are a topical source of non-invasive biomarkers for health and diseases of the urogenital system. However, several challenges have become evident in the standardization of uEV pipelines from collection of urine to biomarker analysis. Here, we studied the effect of pre-analytical variables and developed means of quality control for uEV isolates to be used in transcriptomic biomarker research. We included urine samples from healthy controls and individuals with type 1 or type 2 diabetes and normo-, micro-or macroalbuminuria and isolated uEV byThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Exploration of Extracellular Vesicle miRNAs, Targeted mRNAs and Pathways in Prostate Cancer: Relation to Disease Status and Progression

“…Thus, we performed a thorough quality control of the uEV from representative individual patient and pooled control samples by EM, NTA, Western blotting and RNA profiling analysis (Figure 2, n = 3-8 individual samples per method). The EM images showed similar small uEV enriched samples as before [4,7,20,30] with variable levels of THP, but no obvious other contaminants such as cellular remnants or intracellular organelles, including mitochondria. By NTA, particle mean (166-189 nm) and mode (118-139) sizes were similar between samples, while concentrations varied almost 10-fold (1.6 × 10 9 -1.1 × 10 10 particles per ml urine), with both the minimum and maximum concentration detected in patient samples.…”

“…The contradictory results could be explained by the different sample type (urine vs. uEV) and compared groups: we compared PCa status and progression groups, while the other study compared PCa to benign prostate hyperplasia patients [13]. The results could equally well differ due to choices concerning any (pre)analytical stepsstandardization of methods is missing, particularly for uEV [5,7,20,24,64].…”

“…For normalization in assessing the miRNA expression, miR-103a-3p, -99b-5p, -151a-3p and let-7b-5p were selected as reference miRNAs based on NormFinder [35] stability values in the miRNAseq (Table S2) as well as through evidence of uEV expression and PCa literature survey (e.g., [7,11,20,[36][37][38][39]) and non-differential expression between groups in first qPCR. Applied calculations were (1) Normalized delta Cq (dCq) = normalizer assays mean Cq − assay Cq, (2) Differences in expression levels = dCq(1) − dCq(2) = ddCq, and (3) Fold change = 2ˆddCq.…”

“…The mRNA sequencing (mRNAseq) was performed as described [20]. Briefly, for sequencing libraries, 400 pg of total RNA were prepared with SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara BIo Inc., Mountain View, CA, USA) and Nextera XT kit (Ilumina Inc., San Diego, CA, USA) according to the manufacturer's protocols.…”

“…Technically, RNA research into uEV is challenging. The best practices in EV pipelines are currently still developing, and they include pre-analytics (sample collection, processing and storage), EV isolation, RNA detection and data normalization/analysis [5,7,20,21]. The International Society of Extracellular Vesicles (ISEV) has launched several standardization efforts to expedite the development, including, e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines, and targeted ISEV position papers [5,22].…”