Quantitative Analysis of Exosomal miRNA via qPCR and Digital PCR

Bellingham, Shayne A.; Shambrook, Mitch; Hill, Andrew F.

doi:10.1007/978-1-4939-6728-5_5

Cited by 34 publications

(31 citation statements)

References 22 publications

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“…Helwa et al isolated EVs from different starting volumes by precipitation and UC and quantified associated miRNAs by droplet digital PCR [23]. Analysing EV miRNAs using PCR-based assays is an important and well-established approach supported by excellent protocols and methods [24–26]. …”

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

196

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

196

165

View full text Add to dashboard Cite

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“…A DNA melting curve was generated to discriminate between specific and non‐specific amplification products. Real‐time qPCR was performed in duplicate for all samples, and mean for each duplicate was calculated and used for further analysis (Bellingham, Shambrook, & Hill, ; Dini, Loux et al, ; Salone & Rederstorff, ; Vandesompele et al, ).…”

Section: Methodsmentioning

confidence: 99%

Extraction of RNA from formalin‐fixed, paraffin‐embedded equine placenta

Dini

Esteller-Vico

Scoggin

et al. 2019

Reprod Domestic Animals

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Contents Archived formalin‐fixed, paraffin‐embedded (FFPE) samples represent a valuable resource for the determination of gene expression for physio/pathological conditions. In the present study, we validated a protocol for the extraction of RNA from FFPE samples collected from healthy and diseased equine placenta. The quality and quantity of the extracted RNA from the FFPE and matching RNAlater™‐preserved samples and expression levels of common housekeeping genes and reference microRNAs were evaluated. Precision of the expression data was evaluated by comparing relative expression of CYP19A1 and HSD3B1 in FFPE and RNAlater™ samples. The median RNA concentration recovered from FFPE samples was 316.8 ng/mm3 of tissue (ranging between 61.6 and 917.4 ng/mm3), average RNA integrity number was 2.3 ± 0.9 (mean ± standard deviation), and 84% of samples had RNA fragments longer than 200 nucleotides (DV200). RNA concentrations and CT values for GAPDH, ACTB, miR‐8908a and miR‐369 in FFPE samples were significantly correlated (r = −0.8, −0.7, −0.4 and −0.4, respectively; p < 0.001). Expression pattern of normalized CYP19A1 and HSD3B1 in paired FFPE and RNAlater™ samples was significantly correlated (r = 0.97 for CYP19A1 and HSD3B1; p < 0.001). This study demonstrates that RNA can be extracted from FFPE equine placental tissue and used for downstream transcriptomic analysis. Similar RNA expression patterns were obtained using RNAlater™ and FFPE tissue samples.

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“…Nanoflow ultrahigh performance liquid chromatography‐electrospray ionization‐tandem mass spectrometry (nUPLC‐ESI‐MS/MS) was subsequently used to examine the collected EV fractions for lipidomic analysis, indicating a 1.5‐ to twofold increase of overall lipid amount in PCa patients with the increase more significant in the smaller fractions than in the larger ones (a 150 nm diameter borderline) . As the unique nature of the EV bilayer confers its RNA molecules with remarkable resistance against degradation by cellular RNAses even after a storage of extended time period, EV‐specific miRNA signatures can be developed via qRT‐PCR and QuantStudio™ (a three‐dimensional real‐time digital PCR system designed by Applied Biosystems) integrated with a 20K Chip . The techniques are recently documented in a protocol that described the full procedure of capturing EV samples from cell culture medium via standard ultracentrifugation or OptiPrep density gradient fractionation, which is also adaptable for analysis of miRNAs upon EV isolation from biofluids including plasma, serum, or urine …”

Section: Technological Advancements In Ev Isolation Content Analysismentioning

confidence: 99%

“…137 As the unique nature of the EV bilayer confers its RNA molecules with remarkable resistance against degradation by cellular RNAses even after a storage of extended time period, EV-specific miRNA signatures can be developed via qRT-PCR and QuantStudio TM (a three-dimensional real-time digital PCR system designed by Applied Biosystems) integrated with a 20K Chip. 138 The techniques are recently documented in a protocol that described the full procedure of capturing EV samples from cell culture medium via standard ultracentrifugation or OptiPrep density gradient fractionation, which is also adaptable for analysis of miRNAs upon EV isolation from biofluids including plasma, serum, or urine. 138 In vivo noninvasive tracking of EVs under preclinical and clinical conditions is specifically important for the design, development, and optimization of EV-based diagnosis, prognosis, and therapies.…”

Section: Analysis and In Vivo Imagingmentioning

confidence: 99%

“…138 The techniques are recently documented in a protocol that described the full procedure of capturing EV samples from cell culture medium via standard ultracentrifugation or OptiPrep density gradient fractionation, which is also adaptable for analysis of miRNAs upon EV isolation from biofluids including plasma, serum, or urine. 138 In vivo noninvasive tracking of EVs under preclinical and clinical conditions is specifically important for the design, development, and optimization of EV-based diagnosis, prognosis, and therapies. The EV-associated molecular imaging techniques comprise two types of EV labeling, namely direct and indirect.…”

Section: Analysis and In Vivo Imagingmentioning

confidence: 99%

See 1 more Smart Citation

Extracellular vesicles in the tumor microenvironment: Therapeutic resistance, clinical biomarkers, and targeting strategies

Han

et al. 2017

Medicinal Research Reviews

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Numerous studies have proved that cell‐nonautonomous regulation of neoplastic cells is a distinctive and essential characteristic of tumorigenesis. Two way communications between the tumor and the stroma, or within the tumor significantly influence disease progression and modify treatment responses. In the tumor microenvironment (TME), malignant cells utilize paracrine signaling initiated by adjacent stromal cells to acquire resistance against multiple types of anticancer therapies, wherein extracellular vesicles (EVs) substantially promote such events. EVs are nanoscaled particles enclosed by phospholipid bilayers, and can mediate intercellular communications between cancerous cells and the adjacent microenvironment to accelerate pathological proceeding. Here we review the most recent studies of EV biology and focus on key cell lineages of the TME and their EV cargoes that are biologically active and responsible for cancer resistance, including proteins, RNAs, and other potentially essential components. Since EVs are emerging as novel but critical elements in establishing and maintaining hallmarks of human cancer, timely and insightful understanding of their molecular properties and functional mechanisms would pave the road for clinical diagnosis, prognosis, and effective targeting in the global landscape of precision medicine. Further, we address the potential of EVs as promising biomarkers in cancer clinics and summarize the technical improvements in EV preparation, analysis, and imaging. We highlight the practical issues that should be exercised with caution to guide the development of targeting agents and therapeutic methodologies to minimize cancer resistance driven by EVs, thereby allowing to effectively control the early steps of disease exacerbation.

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Quantitative Analysis of Exosomal miRNA via qPCR and Digital PCR

Cited by 34 publications

References 22 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

Extraction of RNA from formalin‐fixed, paraffin‐embedded equine placenta

Extracellular vesicles in the tumor microenvironment: Therapeutic resistance, clinical biomarkers, and targeting strategies

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