Analysis of extracellular vesicle mRNA derived from plasma using the nCounter platform.
Sci Rep
; 11(1): 3712, 2021 02 12.
Article
en En
| MEDLINE
| ID: mdl-33580122
ABSTRACT
Extracellular vesicles (EVs) are double-layered phospholipid membrane vesicles that are released by most cells and can mediate intercellular communication through their RNA cargo. In this study, we tested if the NanoString nCounter platform can be used for the analysis of EV-mRNA. We developed and optimized a methodology for EV enrichment, EV-RNA extraction and nCounter analysis. Then, we demonstrated the validity of our workflow by analyzing EV-RNA profiles from the plasma of 19 cancer patients and 10 controls and developing a gene signature to differentiate cancer versus control samples. TRI reagent outperformed automated RNA extraction and, although lower plasma input is feasible, 500 µL provided highest total counts and number of transcripts detected. A 10-cycle pre-amplification followed by DNase treatment yielded reproducible mRNA target detection. However, appropriate probe design to prevent genomic DNA binding is preferred. A gene signature, created using a bioinformatic algorithm, was able to distinguish between control and cancer EV-mRNA profiles with an area under the ROC curve of 0.99. Hence, the nCounter platform can be used to detect mRNA targets and develop gene signatures from plasma-derived EVs.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Análisis Químico de la Sangre
/
ARN Mensajero
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Vesículas Extracelulares
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Neoplasias
Tipo de estudio:
Observational_studies
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Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Sci Rep
Año:
2021
Tipo del documento:
Article
País de afiliación:
España
Pais de publicación:
ENGLAND
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ESCOCIA
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GB
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GREAT BRITAIN
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INGLATERRA
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REINO UNIDO
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SCOTLAND
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UK
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UNITED KINGDOM