Abstract
Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%–65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.
Highlights
Exosomes are extracellular vesicles (EVs) with a diameter of approximately 30–150 nm and a density of 1.13~1.19 g/mL that are secreted from most cells through sorting pathways
When samples were treated with proteinase K (PK), the levels of non-exosomal proteins were drastically reduced, apolipoprotein A-1 (apo A-1) was scantly detected in extracellular vesicles (ELVs) samples treated with 0.5 mg/mL of PK
When samples were treated with 0.5 mg/mL PK, the levels of TSG101 in 20 μg ELV proteins were lower with the SBI than with the other kits, and the levels of Alix and CD63 were comparable with other kits, while the levels of annexin 5 were higher than those with the LT or QG preparation kit
Summary
Exosomes are extracellular vesicles (EVs) with a diameter of approximately 30–150 nm and a density of 1.13~1.19 g/mL that are secreted from most cells through sorting pathways. Various methods, including differential ultracentrifugation (UC), density-gradient ultracentrifugation (DG-UC), size-exclusion chromatography (SEC), and polymer-based precipitation (PP), allow exosome-like extracellular vesicles (ELVs) to be isolated from biologic fluids or cell culture media, several issues remain unresolved, especially in terms of clinical applications, including the aggregation of vesicles, low recovery, necessity of a large sample volume, and contamination with soluble proteins and lipoproteins [2,3]. Co-isolation with ELV-sized lipoproteins or protein precipitates can interfere with the analysis of ELV-associated micro-RNAs (miRNAs), because certain lipoproteins (e.g., high-density lipoprotein (HDL)) may carry miRNAs [4]. ELV-associated miRNAs might be potential biomarkers for disease diagnosis; they could be used to monitor disease progression [18,19,20] or as therapeutic targets [21]
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