Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data
Fuente:
Wiley Open Access
Saved in:
| Main Authors: | , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Artículo Open Access |
| Published: |
Wiley
2024
|
| Subjects: | |
| Online Access: | |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1867005931908235264 |
|---|---|
| author | Cláudio Pinheiro 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 An Hendrix |
| author_facet | Cláudio Pinheiro 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 An Hendrix Cláudio Pinheiro 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 An Hendrix |
| collection | Wiley Open Access |
| contents | Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data Cláudio Pinheiro 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 An Hendrix Journal of Extracellular Vesicles AbstractExtracellular 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. 10.1002/jev2.12421 http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| doi_str_mv | 10.1002/jev2.12421 |
| format | Artículo Open Access |
| id | wiley_oa_10_1002_jev2_12421 |
| institution | Wiley Open Access |
| license_str_mv | http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| publishDate | 2024 |
| publisher | Wiley |
| record_format | wiley_oa |
| spellingShingle | Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data Cláudio Pinheiro 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 An Hendrix Journal of Extracellular Vesicles Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data Cláudio Pinheiro 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 An Hendrix Journal of Extracellular Vesicles AbstractExtracellular 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. 10.1002/jev2.12421 http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| title | Identification and validation of extracellular vesicle reference genes for the normalization of RT‐qPCR data |
| topic | Journal of Extracellular Vesicles |
| url | https://isevjournals.onlinelibrary.wiley.com/doi/10.1002/jev2.12421 |