Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Buschmann D

PROVIDER: S-EPMC5990937 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Buschmann Dominik D Kirchner Benedikt B Hermann Stefanie S Märte Melanie M Wurmser Christine C Brandes Florian F Kotschote Stefan S Bonin Michael M Steinlein Ortrud K OK Pfaffl Michael W MW Schelling Gustav G Reithmair Marlene M

Journal of extracellular vesicles 20180604 1

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 down ...[more]

PMID: 29887978

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Project description:Extracellular vesicles (EVs) have great potential as a source for clinically relevant biomarkers since they can be readily isolated from biofluids and carry microRNA (miRNA), mRNA, and proteins that can reflect disease status. However, the biological and technical variability of EV content is unknown making comparisons between healthy subjects and patients difficult to interpret. In this study, we sought to establish a laboratory and bioinformatics analysis pipeline to analyse the small RNA content within EVs from patient serum that could serve as biomarkers and to assess the biological and technical variability of EV RNA content in healthy individuals. We sequenced EV small RNA from multiple individuals (biological replicates) and sequenced multiple replicates per individual (technical replicates) using the Illumina Truseq protocol. We observed that the replicates of samples clustered by subject indicating that the biological variability (~95%) was greater than the technical variability (~0.50%). We observed that ~30% of the sequencing reads were miRNAs. We evaluated the technical parameters of sequencing by spiking the EV RNA preparation with a mix of synthetic small RNA and demonstrated a disconnect between input concentration of the spike-in RNA and sequencing read frequencies indicating that bias was introduced during library preparation. To determine whether there are differences between library preparation platforms, we compared the Truseq with the Nextflex protocol that had been designed to reduce bias in library preparation. While both methods were technically robust, the Nextflex protocol reduced the bias and exhibited a linear range across input concentrations of the synthetic spike-ins. Altogether, our results indicate that technical variability is much smaller than biological variability supporting the use of EV small RNAs as potential biomarkers. Our findings also indicate that the choice of library preparation method leads to artificial differences in the datasets generated invalidating the comparability of sequencing data across library preparation platforms.

Dataset Information

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

Publications

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

Similar Datasets

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