Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine

Crossland, Rachel E; Norden, Jean; Bibby, L.; Davis, Joanna; Dickinson, AM

doi:10.1016/j.jim.2015.12.011

Cited by 66 publications

(56 citation statements)

References 38 publications

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“…Given that precipitation is time efficient, inexpensive and demands no specialized equipment, it also seems to conveniently lend itself to integration into clinical usage. However, in a research field as vibrant and international, standardizing reagents and protocols utilized for EV precipitation and characterization are crucial for generating valid and reproducible data across laboratories [22,85,91]. …”

Section: Discussionmentioning

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

198

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: Discussionmentioning

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

198

165

View full text Add to dashboard Cite

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“…Plasma exosomes were extracted using an exoRNeasy kit [27] (QIAGEN GmbH, Hilden, Germany). A quantity of 1 mL of plasma was removed from –80°C storage, thawed on ice, and centrifuged at 16,000 g for 10 min at 4°C to remove cellular materials and coagulated proteins, after which 700 μL of supernatant was transferred into a new tube by careful aspiration.…”

Section: Methodsmentioning

confidence: 99%

“…U6 small nuclear RNA was used for normalization as an internal control [27]. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed using a Roche LightCycler 480 (Roche Applied Science, Germany).…”

Section: Methodsmentioning

confidence: 99%

Diagnosis of Hyperacute and Acute Ischaemic Stroke: The Potential Utility of Exosomal MicroRNA-21-5p and MicroRNA-30a-5p

Wang

et al. 2018

Cerebrovasc Dis

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Background: Early and accurate diagnosis of ischaemic stroke (IS) requires the use of an optimized biomarker. Exosomal microRNAs have the potential to serve as biomarkers owing to their stability and specificity. We investigated the expression levels of plasma-derived exosomal microRNA-21-5p and microRNA-30a-5p in the different phases of IS. Methods: One hundred forty-three patients with IS and 24 non-stroke controls were enrolled. The patients were divided into the following 5 groups: 1 group for the hyperacute phase IS (HIS, within 6 h); two for the acute phase IS (AIS, including days 1–3 and days 4–7); one for the subacute phase IS (SIS, days 8–14); and one for the recovery phase IS (RIS, days >14). Plasma exosomes were isolated using a QIAGEN exoRNeasy kit and examined by transmission electron microscopy, nanoparticle tracking, and flow cytometry. The expression levels of miRNA-21-5p and miRNA-30a-5p were detected by quantitative real-time polymerase chain reaction. Results: The plasma exosomal miR-21-5p levels in SIS and RIS were significantly higher than that in controls (p < 0.05 and p < 0.01 respectively). The levels of miR-30a-5p in HIS were significantly higher (p < 0.05) and in AIS (days 1–3) were lower than that in controls (p < 0.05). In AIS (days 1–3), both miRNAs were decreased compared with the HIS group (p = 0.053 and 0.001, respectively). The area under the curve (AUC) of the miR-21-5p was 0.714 for SIS (95% CI 0.570–0.859, p = 0.007), 0.734 for RIS (95% CI 0.596–0.871, p = 0.003); the AUC of the miR-30a-5p was 0.826 for HIS (95% CI 0.665–0.988, p = 0.001), 0.438 for AIS (days 1–3; 95% CI 0.240–0.635, p = 0.516). Conclusions: The plasma-derived exosomal miR-21-5p and miRNA-30a-5p in combination are promising biomarkers for diagnosing IS and distinguishing among HIS, SIS, and RIS, especially miRNA-30a-5p for the diagnosis of the HIS phase. Our results provide a new reference for clinicians to apply in early-stage diagnosis and identifies the possible value of biomarkers for IS thrombolysis therapy.

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“…Although in this study, we did not use an internal control for calibration of qRT-PCR results, it is important to mention that an appropriate internal control for exRNA studies is currently controversial. Several helpful studies have examined this controversy and may be of use to those working in this field (27–29). …”

Section: Methodsmentioning

confidence: 99%

A method for extracting and characterizing RNA from urine: For downstream PCR and RNAseq analysis

Zhou

Spillman

Behbakht

et al. 2017

Analytical Biochemistry

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Readily accessible samples such as urine or blood are seemingly ideal for differentiating and stratifying patients; however, it has proven a daunting task to identify reliable biomarkers in such samples. Noncoding RNA holds great promise as a source of biomarkers distinguishing physiologic wellbeing or illness. Current methods to isolate and characterize RNA molecules in urine are limited. In this proof of concept study, we present a method to extract and identify small noncoding RNAs in urine. Initially, quantitative reverse transcription PCR was applied to confirm the presence of microRNAs in total RNA extracted from urine. Once the presence of micro RNA in urine was confirmed, we developed a method to scale up RNA extraction to provide adequate amounts of RNA for next generation sequence analysis. The method described in this study is applicable to detecting a broad range of small noncoding RNAs in urine; thus, they have wide applicability for health and disease analyses.

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Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine

Cited by 66 publications

References 38 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

Diagnosis of Hyperacute and Acute Ischaemic Stroke: The Potential Utility of Exosomal MicroRNA-21-5p and MicroRNA-30a-5p

A method for extracting and characterizing RNA from urine: For downstream PCR and RNAseq analysis

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