Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers

Li, Mu; Zeringer, Emily; Barta, Timothy; Schageman, Jeoffrey; Cheng, Angie; Vlassov, Alexander V.

doi:10.1098/rstb.2013.0502

Cited by 334 publications

(312 citation statements)

References 29 publications

(43 reference statements)

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“…Several previous publications reported the feasibility and utility of sequencing small RNA in EVs isolated from serum, plasma, urine, and cell culture supernatant [2,27–30]. Small RNA-Seq experiments often focus on valuable applications such as liquid biopsy-based diagnostics and, consequently, clinical samples.…”

Section: Introductionmentioning

confidence: 99%

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

Buschmann

Kirchner

Hermann

et al. 2018

J of Extracellular Vesicle

197

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: Introductionmentioning

confidence: 99%

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

Buschmann

Kirchner

Hermann

et al. 2018

J of Extracellular Vesicle

197

165

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“…blood, salvia, urine, breast milk, ascites, etc.). [9][10][11] Currently, most exosome based diagnostic approaches focus on identifying specific molecular components by elaborate 'omics' studies. [12] Examples are elevated levels of miR-21 in exosomes of hepatocellular cancer patients [13] and the presence of EGFRvIII mutant proteins on exosomes derived from a specific glioblastoma subtype.…”

Section: Introductionmentioning

confidence: 99%

Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy

Stremersch

Marro

Pinchasik

et al. 2016

Small

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Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer in an early stage. In this study the feasibility of using a Surface Enhanced Raman Spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nano-coating allows the recording of SERS spectra from single vesicles. By using Partial Least Square Discriminative Analysis (PLS-DA) on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for non-invasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis.

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“…These include RNA and proteins; however, the average low lumen size of EVs limits the amount of material that they can carry [5,6]. This limited cargo capacity has proven to be a challenge for capitalizing on the potential of exploiting EVs, as it increases the difficulty in identifying low-abundance material, or increases the demands on limited sample amounts.…”

Section: Introductionmentioning

confidence: 99%

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Joy

Ayre

Chute

et al. 2018

J of Extracellular Vesicle

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Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

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Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers

Cited by 334 publications

References 29 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

Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

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