Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data.

Autor: Pinheiro C; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium., Guilbert N; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium., Lippens L; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium., Roux Q; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium., Boiy R; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium., Fischer S; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium., Van Dorpe S; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium.; Department of Gynecology, Ghent University Hospital, Ghent, Belgium., De Craene B; Cancer Research Institute Ghent (CRIG), Ghent, Belgium.; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium., Berx G; Cancer Research Institute Ghent (CRIG), Ghent, Belgium.; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium., Boterberg T; Department of Radiation Oncology, Ghent University Hospital, Ghent, Belgium., Sys G; Department of Orthopedics and Traumatology, Ghent University Hospital, Ghent, Belgium., Denys H; Cancer Research Institute Ghent (CRIG), Ghent, Belgium.; Department of Internal Medicine and Pediatrics, Medical Oncology, Ghent University Hospital, Ghent, Belgium., Miinalainen I; Biocenter Oulu, University of Oulu, Oulu, Finland., Mestdagh P; Cancer Research Institute Ghent (CRIG), Ghent, Belgium.; OncoRNALab, Department of Biomolecular Medicine, Ghent, Belgium., Vandesompele J; Cancer Research Institute Ghent (CRIG), Ghent, Belgium.; OncoRNALab, Department of Biomolecular Medicine, Ghent, Belgium., De Wever O; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium., Hendrix A; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), Ghent, Belgium.
Jazyk: angličtina
Zdroj: Journal of extracellular vesicles [J Extracell Vesicles] 2024 Apr; Vol. 13 (4), pp. e12421.
DOI: 10.1002/jev2.12421
Abstrakt: Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications.
(© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)
Databáze: MEDLINE
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