Polyethylene glycol improves current methods for circulating extracellular vesicle-derived DNA isolation

Abstract

BackgroundExtracellular vesicles (EVs) are small membrane-bound vesicles which play an important role in cell-to-cell communication. Their molecular cargo analysis is presented as a new source for biomarker detection, and it might provide an alternative to traditional solid biopsies. However, the most effective approach for EV isolation is not yet well established.ResultsHere, we study the efficiency of the most common EV isolation methods-ultracentrifugation, Polyethlyene glycol and two commercial kits, Exoquick® and PureExo®. We isolated circulating EVs from the bloodstream of healthy donors, characterized the size and yield of EVs and analyzed their protein profiles and concentration. Moreover, we have used for the first time Digital-PCR to identify and detect specific gDNA sequences, which has several implications for diagnostic and monitoring many types of diseases.ConclusionsOur findings present Polyethylene glycol precipitation as the most feasible and less cost-consuming EV isolation technique.

Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid

“…This situation is not particularly satisfactory when the amount of biological material that can be retrieved through bronchial washing is limited, as normally applies to newborn and infant cohorts. Polyethylene-glycol (PEG)-based methods have successfully been used to improve the efficiency of recovering bacteriophages, exosomes or edible nanoparticles ( Castro-Mejia et al, 2015 ; Deregibus et al, 2016 ; Garcia-Romero et al, 2019 ; Kalarikkal et al, 2020 ). These methods have also been shown to improve the yield and purity of microbial DNA recovered from different low-biomass ecological niches ( Arbeli and Fuentes, 2007 ).…”

Exosomes and extracellular vesicles as liquid biopsy biomarkers in diffuse large B‐cell lymphoma: Current state of the art and unmet clinical needs

“…89 It is a relatively simple, cost-effective and rapid technique that produces a high yield and preserves the biophysical properties of isolated exosomes. [90][91][92][93][94] Protein removal kits are used to reduce the amount of co-precipitated proteins but protein aggregates are still a concern with the use of polymer-based precipitation. 95,96 Immunoaffinity enrichment methods use antibodies to exosomal membrane markers, typically the tetraspanins CD9, CD63 and CD81, to isolate exosomes from biofluids.…”

“…In polymer‐based precipitation, a polymer, eg, polyethylene glycol (PEG), precipitates exosomes in a biofluid via dehydration, and the exosomes are recovered by low‐speed centrifugation to pellet down the precipitate 89 . It is a relatively simple, cost‐effective and rapid technique that produces a high yield and preserves the biophysical properties of isolated exosomes 90–94 . Protein removal kits are used to reduce the amount of co‐precipitated proteins but protein aggregates are still a concern with the use of polymer‐based precipitation 95,96 …”

Microfluidic‐Based Exosome Analysis for Liquid Biopsy

“…Highly hydrophilic polymers like polyethylene glycol (PEG) interact with water molecules and create a hydrophobic micro‐environment around the exosomes, facilitating their precipitation. [ 62 ] However, other water‐soluble materials can co‐precipitate, resulting in significant contamination. [ 57,63 ] The immunoaffinity‐based isolation method uses beads modified with antibodies toward specific biomarkers such as CD63, CD81, and CD9 for exosome capture.…”