RNA-Seq is not required to determine stable reference genes for qPCR normalization.
Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. Hence, recent...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2022-02-01
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| Series: | PLoS Computational Biology |
| Online Access: | https://doi.org/10.1371/journal.pcbi.1009868 |
| _version_ | 1818475021642760192 |
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| author | Nirmal Kumar Sampathkumar Venkat Krishnan Sundaram Prakroothi S Danthi Rasha Barakat Shiden Solomon Mrityunjoy Mondal Ivo Carre Tatiana El Jalkh Aïda Padilla-Ferrer Julien Grenier Charbel Massaad Jacqueline C Mitchell |
| author_facet | Nirmal Kumar Sampathkumar Venkat Krishnan Sundaram Prakroothi S Danthi Rasha Barakat Shiden Solomon Mrityunjoy Mondal Ivo Carre Tatiana El Jalkh Aïda Padilla-Ferrer Julien Grenier Charbel Massaad Jacqueline C Mitchell |
| author_sort | Nirmal Kumar Sampathkumar |
| collection | DOAJ |
| description | Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization. In this study, however, we demonstrate that the statistical approach to determine the best reference genes from commonly used conventional candidates is more important than the preselection of 'stable' candidates from RNA-Seq data. Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using conventional reference genes render the same results as stable reference genes selected from RNA-Seq data. We validated these observations in two distinct cross-sectional experimental conditions involving human iPSC derived microglial cells and mouse sciatic nerves. These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays. |
| first_indexed | 2024-04-14T04:44:05Z |
| format | Article |
| id | doaj.art-e62e27386f794ee6b92abeaa7293f8bd |
| institution | Directory Open Access Journal |
| issn | 1553-734X 1553-7358 |
| language | English |
| last_indexed | 2024-04-14T04:44:05Z |
| publishDate | 2022-02-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Computational Biology |
| spelling | doaj.art-e62e27386f794ee6b92abeaa7293f8bd2022-12-22T02:11:31ZengPublic Library of Science (PLoS)PLoS Computational Biology1553-734X1553-73582022-02-01182e100986810.1371/journal.pcbi.1009868RNA-Seq is not required to determine stable reference genes for qPCR normalization.Nirmal Kumar SampathkumarVenkat Krishnan SundaramPrakroothi S DanthiRasha BarakatShiden SolomonMrityunjoy MondalIvo CarreTatiana El JalkhAïda Padilla-FerrerJulien GrenierCharbel MassaadJacqueline C MitchellAssessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization. In this study, however, we demonstrate that the statistical approach to determine the best reference genes from commonly used conventional candidates is more important than the preselection of 'stable' candidates from RNA-Seq data. Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using conventional reference genes render the same results as stable reference genes selected from RNA-Seq data. We validated these observations in two distinct cross-sectional experimental conditions involving human iPSC derived microglial cells and mouse sciatic nerves. These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays.https://doi.org/10.1371/journal.pcbi.1009868 |
| spellingShingle | Nirmal Kumar Sampathkumar Venkat Krishnan Sundaram Prakroothi S Danthi Rasha Barakat Shiden Solomon Mrityunjoy Mondal Ivo Carre Tatiana El Jalkh Aïda Padilla-Ferrer Julien Grenier Charbel Massaad Jacqueline C Mitchell RNA-Seq is not required to determine stable reference genes for qPCR normalization. PLoS Computational Biology |
| title | RNA-Seq is not required to determine stable reference genes for qPCR normalization. |
| title_full | RNA-Seq is not required to determine stable reference genes for qPCR normalization. |
| title_fullStr | RNA-Seq is not required to determine stable reference genes for qPCR normalization. |
| title_full_unstemmed | RNA-Seq is not required to determine stable reference genes for qPCR normalization. |
| title_short | RNA-Seq is not required to determine stable reference genes for qPCR normalization. |
| title_sort | rna seq is not required to determine stable reference genes for qpcr normalization |
| url | https://doi.org/10.1371/journal.pcbi.1009868 |
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