Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by Next‐Generation Sequencing

Buschmann, Dominik; Kirchner, Benedikt; Hermann, Stefanie; Märte, Melanie; Wurmser, Christine; Brandes, Florian; Kotschote, Stefan; Bonin, Michael; Steinlein, Ortrud K.; Pfaffl, Michael W.; Schelling, Gustav; Reithmair, Marlene

doi:10.1080/20013078.2019.1581487

Cited by 27 publications

(36 citation statements)

References 1 publication

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“…EV separation is the foundation of EV work, determining what material will be further analyzed. Separation methods are based on different principles that may provide EV products containing distinct EV subpopulations with different levels of contaminants (Buschmann et al., 2018, Royo et al., 2016). The most commonly used EV separation methods are ultracentrifugation (UC), size exclusion chromatography (SEC) and polymer‐based precipitation (Gardiner et al., 2016).…”

Section: Introductionmentioning

confidence: 99%

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

Dong

Zieren

Horie

et al. 2020

J of Extracellular Vesicle

144

142

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One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

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Section: Introductionmentioning

confidence: 99%

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

Dong

Zieren

Horie

et al. 2020

J of Extracellular Vesicle

144

142

View full text Add to dashboard Cite

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“…www.nature.com/scientificreports www.nature.com/scientificreports/ (comparable to the method used in our studies) and membrane affinity are suitable for miRNA discovery in exosomes 4 . Overall, challenges in isolating exosomes include reproducibility and consistency between techniques.…”

Section: Mrna -Genementioning

confidence: 62%

“…This is further compounded by the fact that a single cancer type may be heterogeneous, presumably due to disparate genetic defects in their tumors 1 . Recently, studies have been directed towards identification of biomarkers for cancer and other diseases, through non-invasive means utilizing components in blood such as circulating tumor cells (CTCs), cell free DNA (cfDNA) and very recently extracellular vesicles (EVs) which includes microvesicles and exosomes [2][3][4] . These liquid biopsy (noninvasive) based analysis present an alternate to conventional tumor biopsies (invasive) and may facilitate biomarker identification to detect early stages of a disease.…”

mentioning

confidence: 99%

“…EVs such as exosomes are released into blood circulation and may have important functions in physiological and pathological conditions. These vesicles have recently invoked interest due to their potential in disease diagnosis and treatment 3,4 . They originate from a multivesicular body and upon fusing with the plasma membrane of the cell, they are released into the extracellular environment 5 .…”

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confidence: 99%

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RNA cargos in extracellular vesicles derived from blood serum in pancreas associated conditions

Kumar

Kimchi

Manjunath

et al. 2020

Sci Rep

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Exosomes are extracellular vesicles which are released from healthy and tumor cells into blood circulation. Unique biomolecular cargos such as RnA and protein are loaded in these vesicles. these molecules may have biological functions such as signaling, cell communications and have the potential to be analyzed as biomarkers. In this initial study, we describe the analysis of exosomes in the serum of healthy subjects, intraductal papillary mucosal neoplasms and pancreatic ductal adenocarcinoma including the characterization of their RNA cargos by next generation sequencing (EXO-NGS). Results indicate the presence of a wide variety of RNAs including mRNA, miRNA, lincRNA, tRNA and piRNA in these vesicles. Based on the differential mRNA expression observed upon EXO-NGS analysis, we independently evaluated two protein coding genes, matrix metalloproteinase-8 (MMP-8) and transcription factor T-Box 3 (TBX3) by qRT-PCR for selective expression in the serum samples. Results indicate a variable expression pattern of these genes across serum samples between different study groups. Further, qRT-PCR analysis with the same serum exosomes processed for EXO-NGS, we observed two long non-coding RNAs, malat-1 and CRNDE to be variably expressed. Overall, our observations emphasize the potential value of different exosome components in distinguishing between healthy, premalignant and malignant conditions related to the pancreas.

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“…Albumin forms a complex network with multiple proteins, lipids and peptide hormones, known as the 'albuminome' [36,37], making its selective, specific depletion challenging. In addition to albumin, other highly-abundant blood proteins such as haemoglobin, serotransferrin, complement and immunoglobulins commonly isolate with blood-EVs [38]. These highly-abundant proteins mask the detection of less-abundant, potential EV biomarker proteins in traditional shot-gun MS analyses [39] based on information dependent acquisition (IDA) methods [40].…”

Section: Introductionmentioning

confidence: 99%

A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

Hallal

Azimi

Wei

et al. 2020

Preprint

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There is a real need for biomarkers that can indicate glioma disease burden and inform clinical management, particularly in the recurrent glioblastoma (GBM; grade IV glioma) setting where treatment-associated brain changes can confound current and expensive tumour surveillance methods. In this regard, extracellular vesicles (EVs; 30-1000 nm membranous particles) hold major promise as robust tumour biomarkers. GBM-EVs encapsulate molecules that reflect the identity and molecular state of their cell-of-origin and cross the blood-brain-barrier into the periphery where they are readily accessible. Despite the suitability of circulating-EVs for GBM biomarker discovery, sample complexity has hindered comprehensive quantitative proteomic studies. Here, sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used in conjunction with a targeted data extraction strategy to comprehensively profile circulating-EVs isolated from plasma. Plasma-EVs sourced from pre-operative glioma II-IV patients (n=41) and controls (n=11) were sequenced by SWATH-MS, and the identities and absolute quantities of the proteins were extracted by aligning the SWATH-MS data against a custom glioma spectral library comprised of 8662 high confidence protein species. Overall, 4054 plasma-EV proteins were quantified across the cohorts, and putative circulating-EV biomarker proteins identified (adjusted p-value<0.05) included previously reported GBM-EV proteins identified in vitro and in neurosurgical aspirates. Principle component analyses showed that plasma-EV protein profiles clustered according to glioma subtype and WHO-grade, and plasma-EV proteins reflected the extent of glioma aggression. Using SWATH-MS, we describe the most comprehensive proteomic plasma-EV profiles for glioma and highlight the promise of this approach as an accurate and sensitive tumour monitoring method. Objective blood-based measurements of glioma tumour activity will support the implementation of next-generation, patient-centred therapies and are ideal surrogate endpoints for recurrent progression that would allow clinical trial protocols to be more dynamic and adapt to the individual patient and their cancer.

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Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by Next‐Generation Sequencing

Cited by 27 publications

References 1 publication

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

RNA cargos in extracellular vesicles derived from blood serum in pancreas associated conditions

A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

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