Isolation of Extracellular Vesicles from Biological Fluids via the Aggregation–Precipitation Approach for Downstream miRNAs Detection

Abstract

Extracellular vesicles (EVs) have high potential as sources of biomarkers for non-invasive diagnostics. Thus, a simple and productive method of EV isolation is demanded for certain scientific and medical applications of EVs. Here we aim to develop a simple and effective method of EV isolation from different biofluids, suitable for both scientific, and clinical analyses of miRNAs transported by EVs. The proposed aggregation–precipitation method is based on the aggregation of EVs using dextran blue and the subsequent precipitation of EVs using 1.5% polyethylene glycol solutions. The developed method allows the effective isolation of EVs from plasma and urine. As shown using TEM, dynamic light scattering, and miRNA analyses, this method is not inferior to ultracentrifugation-based EV isolation in terms of its efficacy, lack of inhibitors for polymerase reactions and applicable for both healthy donors and cancer patients. This method is fast, simple, does not need complicated equipment, can be adapted for different biofluids, and has a low cost. The aggregation–precipitation method of EV isolation accessible and suitable for both research and clinical laboratories. This method has the potential to increase the diagnostic and prognostic utilization of EVs and miRNA-based diagnostics of urogenital pathologies.

Principles and Problems of Exosome Isolation from Biological Fluids

“…A variant of optimization the PEG precipitation technique that allows obtaining more "pure" exosome preparations is described in [55]. The authors suggested enriching the sample with membrane vesicles before adding PEG.…”

The Role of Extracellular Vesicles as Shuttles of RNA and Their Clinical Significance as Biomarkers in Hepatocellular Carcinoma

“…Precipitation-based isolation is inexpensive, requires no special equipment, and is compatible with both low-and highsample volumes. Nevertheless, this method suffers from co-isolation of non-EV particles and protein complexes and must be further improved [57].…”

Extracellular Vesicles: New Tools for Early Diagnosis of Breast and Genitourinary Cancers

“…EVs that are regularly secreted by normal cells into the extracellular space can be recovered in many if not all biological fluids [16,17]. More importantly, specific cancer-cellderived EVs are released into the extracellular space and can be found in plasma, serum, and urine of tumor-bearing patients.…”

“…Ultracentrifugation (UC), density gradient centrifugation (DC), precipitation, immunoaffinity capture, size exclusion chromatography (SEC), and microfluidic-based isolation are the most relevant EV isolation procedures for patients with suspected cancer [17,18]. UC has historically been regarded as the gold standard for EV isolation in basic and translational research.…”

“…In addition, Konoshenko et al [17] proposed a precipitation-based isolation protocol from blood and urine in healthy and prostate cancer patients requiring no more than 110 min for sample preparation and EV isolation. Considering that one of the most commonly ordered diagnostic tests, the blood culture test, generally requires a turn-around time (TAT) of 2 days, we are confident that EVs can become good candidates to enter into clinical practice as diagnostic tools [180].…”