Comparative analysis of EV isolation procedures for miRNAs detection in serum samples

Andreu, Zoraida; Rivas, Eva; Sanguino-Pascual, Aitana; Lamana, Amalia; Marazuela, Mónica; González‐Álvaro, Isidoro; Sánchez-Madrid, Francisco; Fuente, Hortensia de la; Yáñez-Mó, Marı́a

doi:10.3402/jev.v5.31655

Cited by 150 publications

(122 citation statements)

References 27 publications

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“…In line with this, we report precipitation-based isolation to yield samples with lower purity and significant protein contamination, but excellent potential for transcriptomics-driven biomarker discovery. We agree with previous publications stating that enriching serum EVs by precipitation might be a viable strategy for biomarker discovery studies [21,23]. Alvarez et al presented similar findings for profiling RNA biomarkers in urinary EVs [9].…”

Section: Discussionsupporting

confidence: 92%

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

Buschmann

Kirchner

Hermann

et al. 2018

J of Extracellular Vesicle

203

165

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Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate EVs from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications. To help clinicians and scientists choose the most appropriate approach for their experiments, isolation methods need to be comparatively characterized. Few attempts have been made to comprehensively analyse vesicular microRNAs (miRNAs) in patient biofluids for biomarker studies. To address this discrepancy, we set out to benchmark the performance of several isolation principles for serum EVs in healthy individuals and critically ill patients. Here, we compared five different methods of EV isolation in combination with two RNA extraction methods regarding their suitability for biomarker discovery-focused miRNA sequencing as well as biological characteristics of captured vesicles. Our findings reveal striking method-specific differences in both the properties of isolated vesicles and the ability of associated miRNAs to serve in biomarker research. While isolation by precipitation and membrane affinity was highly suitable for miRNA-based biomarker discovery, methods based on size-exclusion chromatography failed to separate patients from healthy volunteers. Isolated vesicles differed in size, quantity, purity and composition, indicating that each method captured distinctive populations of EVs as well as additional contaminants. Even though the focus of this work was on transcriptomic profiling of EV-miRNAs, our insights also apply to additional areas of research. We provide guidance for navigating the multitude of EV isolation methods available today and help researchers and clinicians make an informed choice about which strategy to use for experiments involving critically ill patients.

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Section: Discussionsupporting

confidence: 92%

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

Buschmann

Kirchner

Hermann

et al. 2018

J of Extracellular Vesicle

203

165

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“…This impact on exosome recovery in UC is congruent with the lower total number of particles seen in our UC samples compared to the kits, a trend which has also been previously observed by another group [38]. The more particles from the commercial reagents might be because PEG (polyethylene glycol)-based volume exclusion is used to precipitate non-exosome nanoparticles, such as serum protein aggregates, as supported by the lower exosome purity reported in a recent method-development study of ExtraPEG [41, 62]. …”

Section: Discussionsupporting

confidence: 89%

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

et al. 2017

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Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40–150 nm). The three kits, though, produced a significantly higher yield (80–300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.

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“…The RNA sequencing analysis revealed that miRNAs were enriched in UEVs . However, the EVs RNA patterns showed differ significantly in respect to the method used for RNA extraction . Thus, the RNA quantity and RNA distribution were measured in our study.…”

Section: Discussionmentioning

confidence: 86%

MiR‐145 detection in urinary extracellular vesicles increase diagnostic efficiency of prostate cancer based on hydrostatic filtration dialysis method

Qin

et al. 2017

The Prostate

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Comparative analysis of EV isolation procedures for miRNAs detection in serum samples

Cited by 150 publications

References 27 publications

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

MiR‐145 detection in urinary extracellular vesicles increase diagnostic efficiency of prostate cancer based on hydrostatic filtration dialysis method

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