Analysis of extracellular vesicle mRNA derived from plasma using the nCounter platform
Por:
Bracht, JWP, Gimenez-Capitan, A, Huang, CY, Potie, N, Pedraz-Valdunciel, C, Warren, S, Rosell, R and Molina-Vila, MA
Publicada:
12 feb 2021
Ahead of Print:
12 feb 2021
Resumen:
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 mu L 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.
Filiaciones:
Bracht, JWP:
Quiron Dexeus Univ Hosp, Lab Oncol, Pangaea Oncol, Sabino Arana 5-19, Barcelona 08028, Spain
Univ Autonoma Barcelona UAB, Dept Biochem Mol Biol & Biomed, Cerdanyola Del Valles 08193, Spain
Gimenez-Capitan, A:
Quiron Dexeus Univ Hosp, Lab Oncol, Pangaea Oncol, Sabino Arana 5-19, Barcelona 08028, Spain
Huang, CY:
NanoString Technol, Seattle, WA USA
Potie, N:
Univ Granada, Dept Genet, Fac Sci, Granada 18071, Spain
PTS, Biotechnol Inst, Ctr Invest Biomed, Bioinformat Lab, Avda Conocimiento S-N, Granada 18100, Spain
:
Univ Autonoma Barcelona UAB, Dept Biochem Mol Biol & Biomed, Cerdanyola Del Valles 08193, Spain
Germans Trias & Pujol Hlth Sci Inst & Hosp IGTP, Barcelona, Spain
Warren, S:
NanoString Technol, Seattle, WA USA
:
Germans Trias & Pujol Hlth Sci Inst & Hosp IGTP, Barcelona, Spain
Molina-Vila, MA:
Quiron Dexeus Univ Hosp, Lab Oncol, Pangaea Oncol, Sabino Arana 5-19, Barcelona 08028, Spain
Green Published, gold
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