Comparison of EVs isolation methods for miRNAs sequencing
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ABSTRACT:
PROVIDER: PRJNA928492 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23249719.fastq.gz | Fastqsanger.gz | |||
| SRR23249720.fastq.gz | Fastqsanger.gz | |||
| SRR23249721.fastq.gz | Fastqsanger.gz | |||
| SRR23249722.fastq.gz | Fastqsanger.gz | |||
| SRR23249723.fastq.gz | Fastqsanger.gz |
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Project description:Background & aims: MicroRNAs (miRNAs) encapsulated in EVs are potential diagnostic and prognostic biomarkers. However, discrepancies on miRNA patterns and their validation are still frequent due to differences in sample origin, EVs isolation, miRNA extraction and sequencing methods. Selecting appropriate EVs isolation methods is therefore a critical step for miRNA-based biomarker discovery. The aim of the present study is to find the most suitable EVs isolation method for miRNAs sequencing adequate for clinical application. Material & Methods EVs were isolated by Size Exclusion Chromatography (SEC), iodixanol gradients (GRAD) and the combination of both (SEC+GRAD), using the same plasma sample, in triplicate isolation assays. Isolated EVs were characterized and RNA was extracted. Three different protocols for miRNA library preparation were compared (NEBNext, NEXTFlex and SMARTer smRNA-seq) and miRNAs encapsulated on EVs were sequenced using NextSeq 500 system (Illumina). Finally, the yield, abundance and diversity of miRNAs using the three different EVs isolation protocols were analyzed and compared between them. Results The majority of lipoproteins, total cholesterol and plasma proteins were removed from the EVs-containing fractions by using SEC, GRAD, and SEC+GRAD. SEC method recovered a larger amount of EVs followed by GRAD and SEC+GRAD, while GRAD and SEC+GRAD yielded the purest vesicles. NEBNext was the library preparation kit that showed the highest reproducibility among replicas, higher number of reads corresponding to miRNAs and more different miRNAs, followed by NEXTFlex and SMARTer smRNA-seq. GRAD method showed the highest reproducibility among replicas, a higher number of reads corresponding to miRNAs and more different miRNAs, followed by SEC and SEC+GRAD methods. Conclusions These results render the GRAD method to isolate EVs as one of the most appropriate to detect miRNAs from Evs.
2023-04-26 | GSE223838 | GEO
Project description:Salivary extracellular vesicles (EVs) represent a powerful, non-invasive source of biomarkers for disease diagnosis and monitoring. Their molecular cargo reflects systemic and local physiological states, offering a window into neurological and inflammatory disorders. However, the diversity of EV isolation protocols and the possibility that each enriches distinct EV subpopulations remains a major barrier to reproducibility and data comparability. We conducted a comprehensive comparison of three EV isolation methods: ultracentrifugation (UC), PEG-based precipitation (Q), and immunoaffinity capture (M) to evaluate their impact on EV yield, purity, and molecular composition. Salivary EVs from healthy volunteers were analysed using proteomic and small-RNA sequencing approaches. Principal component analysis revealed clear isolation method-dependent clustering, where M-derived EVs displayed the most distinct profile. UC and Q produced broader proteomic repertoires with higher total protein content, whereas M-isolated EVs exhibited greater purity and enrichment of trafficking- and lysosome-associated proteins. Over 731 miRNAs selected, 28 were consistently altered across methods and 65 uniquely enriched in M isolates. RT-qPCR confirmed key directional trends. These 93 method-dependent miRNAs have predicted targets associated with synaptic structure and neurodegenerative pathways. These findings show that isolation methodology deeply shapes salivary EV cargo and suggest that immunoaffinity capture can isolate specific EV populations meeting diagnostic requirements.
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Project description:Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, including miRNAs. The optimal isolation method of uEVs for miRNA profiling in patients with proteinuria is unknown. Six different methods were compared for the isolation of microvesicles and these were compared to raw urine (method A). 2 methods failed and further analysis was performed with the following methods: ExoRNeasy (method C), modified protocol for ExoRNeasy (method E) and ultracentrifugation (method F). Different miRNAs are enriched by method C and F. No differences were observed between method C and E.
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Project description:The lack of standardized protocols for isolating extracellular vesicles (EVs), particularly from biobank-stored plasma, poses a substantial limitation for the study of biomarkers. Combining current isolation methods could enhance the specificity and purity of EVs isolation for their further use in diagnosis and personalized medicine. In this study, plasma samples from healthy individuals were subjected to extracellular vesicle isolation using ultracentrifugation (UC), size exclusion chromatography (SEC), and a combined approach of SEC and UC (SEC+UC). Characterization of the isolated EVs with NTA and TEM indicated that all isolation methods successfully isolated EVs, being not altered due to the long-term storage. Also, the western blot analysis confirmed the presence of exosome markers in all isolates but showed a higher expression of albumin in EVs-UC, suggesting potential plasma protein contamination. Proteomic analysis of samples from different isolation methods identified 542 proteins, with SEC+UC yielding the most complex proteome at 364 proteins. GO analysis of cellular component revealed differences in terms related to EVs and plasma, where SEC+UC proteins had the highest proportion of exosomal proteins. As a result of further study of the unique proteins detected in each isolation method, it was revealed that the SEC+UC method detected 181 unique proteins, demonstrating its superiority in the detection of low plasma concentration proteins. These findings support the robustness of the SEC+UC approach for EV isolation and proteomic studies due to its high efficiency and purity in isolating EVs. This study emphasizes the efficacy of the SEC+UC approach in obtaining highly pure and diverse EV populations suitable for comprehensive proteomic analysis with application to the discovery of biomarkers from biobank-stored plasma.
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2022-02-15 | E-MTAB-11417 | biostudies-arrayexpress
Project description:Cerebrospinal fluid (CSF) miRNAs have emerged as a potential low invasive diagnostic tool for central nervous system malignancies. However, they have not yet been implemented in the clinic since there is no standardized and simple method established. Another problem that it must be considered is that the amount of CSF that can be obtained from patients is very limited, especially from infants. With this in mind, we have compared six different methodologies to establish the best one for the detection of miRNAs using a minimum CSF volume. Four of them incorporated a EVs enrichment step and the other two extracted the miRNAs directly from crude CSF. The efficiency of each method was evaluated by quantitative PCR (qPCR) and smallRNA sequencing. Our results showed that, the Plasma/Serum RNA Purification kit (Norgen) and the proposed method combining the total exosome isolation reagent (Invitrogen) with the mirVana PARIS kit (Ambion) were the most efficient. However, the Norgen’s kit was the most reproducible protocol by qPCR and presented a user-friendly protocol. Therefore, we considered it as the most suitable method for detecting circulating and EVs-associated miRNAs using only 200 µl of CSF.
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