Analysis of extracellular vesicle mRNA derived from plasma using the nCounter platform
Bracht, Jillian (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Giménez-Capitán, Ana (Quirón Dexeus University Hospital. Pangaea Oncology, Laboratory of Oncology)
Huang, Chung-Ying (NanoString Technologies, Seattle, WA USA)
Potie, Nicolas (Biotechnology Institute, Centro de Investigacion Biomedica, PTS. Bioinformatics Laboratory)
Pedraz-Valdunciel, Carlos (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Warren, Sarah (NanoString Technologies, Seattle, WA USA)
Rosell, Rafael (Institut Germans Trias i Pujol. Hospital Universitari Germans Trias i Pujol)
Molina-Vila, Miguel Ángel (Quirón Dexeus University Hospital. Pangaea Oncology, Laboratory of Oncology)

Data:	2021
Resum:	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.
Ajuts:	European Commission 765492
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Gene expression analysis ; Tumour biomarkers ; Biomarkers ; Cancer ; RNA
Publicat a:	Scientific reports, Vol. 11 (february 2021) , ISSN 2045-2322

DOI: 10.1038/s41598-021-83132-0
PMID: 33580122

12 p, 4.7 MB

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