Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data
- Author
- Cláudio Pinheiro (UGent) , Niké Guilbert (UGent) , Lien Lippens (UGent) , Quentin Roux (UGent) , Robin Boiy (UGent) , Suzanne Fischer (UGent) , Sofie Van Dorpe (UGent) , Bram De Craene (UGent) , Geert Berx (UGent) , Tom Boterberg (UGent) , Gwen Sys (UGent) , Hannelore Denys (UGent) , Ilkka Miinalainen, Pieter Mestdagh (UGent) , Jo Vandesompele (UGent) , Olivier De Wever (UGent) and An Hendrix (UGent)
- Organization
- Project
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- TRAIN-EV (Training in Extracellular Vesicles: for benefit in Health and Disease)
- Development and implementation of analytical methods based on laser ablation – and single-event ICP-mass spectrometry for examining metal(loid) signatures in cancer
- Function and biogenesis of extracellular vesicle subsets in cancer
- Abstract
- 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.
- Keywords
- Cell Biology, Histology, urine, tissue, RNase, reference genes, protease, mRNA, microvesicles, extracellular vesicles, exosomes, conditioned medium, blood
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-01HT4J09HHRH8D3ED120JY6F3A
- MLA
- Pinheiro, Cláudio, et al. “Identification and Validation of Extracellular Vesicle Reference Genes for the Normalization of RT‐qPCR Data.” JOURNAL OF EXTRACELLULAR VESICLES, vol. 13, no. 4, 2024, doi:10.1002/jev2.12421.
- APA
- Pinheiro, C., Guilbert, N., Lippens, L., Roux, Q., Boiy, R., Fischer, S., … Hendrix, A. (2024). Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data. JOURNAL OF EXTRACELLULAR VESICLES, 13(4). https://doi.org/10.1002/jev2.12421
- Chicago author-date
- Pinheiro, Cláudio, Niké Guilbert, Lien Lippens, Quentin Roux, Robin Boiy, Suzanne Fischer, Sofie Van Dorpe, et al. 2024. “Identification and Validation of Extracellular Vesicle Reference Genes for the Normalization of RT‐qPCR Data.” JOURNAL OF EXTRACELLULAR VESICLES 13 (4). https://doi.org/10.1002/jev2.12421.
- Chicago author-date (all authors)
- Pinheiro, Cláudio, Niké Guilbert, Lien Lippens, Quentin Roux, Robin Boiy, Suzanne Fischer, Sofie Van Dorpe, Bram De Craene, Geert Berx, Tom Boterberg, Gwen Sys, Hannelore Denys, Ilkka Miinalainen, Pieter Mestdagh, Jo Vandesompele, Olivier De Wever, and An Hendrix. 2024. “Identification and Validation of Extracellular Vesicle Reference Genes for the Normalization of RT‐qPCR Data.” JOURNAL OF EXTRACELLULAR VESICLES 13 (4). doi:10.1002/jev2.12421.
- Vancouver
- 1.Pinheiro C, Guilbert N, Lippens L, Roux Q, Boiy R, Fischer S, et al. Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data. JOURNAL OF EXTRACELLULAR VESICLES. 2024;13(4).
- IEEE
- [1]C. Pinheiro et al., “Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data,” JOURNAL OF EXTRACELLULAR VESICLES, vol. 13, no. 4, 2024.
@article{01HT4J09HHRH8D3ED120JY6F3A,
abstract = {{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.}},
articleno = {{e12421}},
author = {{Pinheiro, Cláudio and Guilbert, Niké and Lippens, Lien and Roux, Quentin and Boiy, Robin and Fischer, Suzanne and Van Dorpe, Sofie and De Craene, Bram and Berx, Geert and Boterberg, Tom and Sys, Gwen and Denys, Hannelore and Miinalainen, Ilkka and Mestdagh, Pieter and Vandesompele, Jo and De Wever, Olivier and Hendrix, An}},
issn = {{2001-3078}},
journal = {{JOURNAL OF EXTRACELLULAR VESICLES}},
keywords = {{Cell Biology,Histology,urine,tissue,RNase,reference genes,protease,mRNA,microvesicles,extracellular vesicles,exosomes,conditioned medium,blood}},
language = {{eng}},
number = {{4}},
pages = {{17}},
title = {{Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data}},
url = {{http://doi.org/10.1002/jev2.12421}},
volume = {{13}},
year = {{2024}},
}
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