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Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a very sensitive widespread technique considered as the gold standard to explore transcriptional variations. While a particular methodology has to be followed to provide accurate results many published studies are li...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385181/ https://www.ncbi.nlm.nih.gov/pubmed/30792499 http://dx.doi.org/10.1038/s41598-019-39072-x |
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author | Koch, Lionel Poyot, Thomas Schnetterle, Marine Guillier, Sophie Soulé, Estelle Nolent, Flora Gorgé, Olivier Neulat-Ripoll, Fabienne Valade, Eric Sebbane, Florent Biot, Fabrice |
author_facet | Koch, Lionel Poyot, Thomas Schnetterle, Marine Guillier, Sophie Soulé, Estelle Nolent, Flora Gorgé, Olivier Neulat-Ripoll, Fabienne Valade, Eric Sebbane, Florent Biot, Fabrice |
author_sort | Koch, Lionel |
collection | PubMed |
description | Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a very sensitive widespread technique considered as the gold standard to explore transcriptional variations. While a particular methodology has to be followed to provide accurate results many published studies are likely to misinterpret results due to lack of minimal quality requirements. Yersinia pestis is a highly pathogenic bacterium responsible for plague. It has been used to propose a ready-to-use and complete approach to mitigate the risk of technical biases in transcriptomic studies. The selection of suitable reference genes (RGs) among 29 candidates was performed using four different methods (GeNorm, NormFinder, BestKeeper and the Delta-Ct method). An overall comprehensive ranking revealed that 12 following candidate RGs are suitable for accurate normalization: gmk, proC, fabD, rpoD, nadB, rho, thrA, ribD, mutL, rpoB, adk and tmk. Some frequently used genes like 16S RNA had even been found as unsuitable to study Y. pestis. This methodology allowed us to demonstrate, under different temperatures and states of growth, significant transcriptional changes of six efflux pumps genes involved in physiological aspects as antimicrobial resistance or virulence. Previous transcriptomic studies done under comparable conditions had not been able to highlight these transcriptional modifications. These results highlight the importance of validating RGs prior to the normalization of transcriptional expression levels of targeted genes. This accurate methodology can be extended to any gene of interest in Y. pestis. More generally, the same workflow can be applied to identify and validate appropriate RGs in other bacteria to study transcriptional variations. |
format | Online Article Text |
id | pubmed-6385181 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-63851812019-02-26 Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions Koch, Lionel Poyot, Thomas Schnetterle, Marine Guillier, Sophie Soulé, Estelle Nolent, Flora Gorgé, Olivier Neulat-Ripoll, Fabienne Valade, Eric Sebbane, Florent Biot, Fabrice Sci Rep Article Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a very sensitive widespread technique considered as the gold standard to explore transcriptional variations. While a particular methodology has to be followed to provide accurate results many published studies are likely to misinterpret results due to lack of minimal quality requirements. Yersinia pestis is a highly pathogenic bacterium responsible for plague. It has been used to propose a ready-to-use and complete approach to mitigate the risk of technical biases in transcriptomic studies. The selection of suitable reference genes (RGs) among 29 candidates was performed using four different methods (GeNorm, NormFinder, BestKeeper and the Delta-Ct method). An overall comprehensive ranking revealed that 12 following candidate RGs are suitable for accurate normalization: gmk, proC, fabD, rpoD, nadB, rho, thrA, ribD, mutL, rpoB, adk and tmk. Some frequently used genes like 16S RNA had even been found as unsuitable to study Y. pestis. This methodology allowed us to demonstrate, under different temperatures and states of growth, significant transcriptional changes of six efflux pumps genes involved in physiological aspects as antimicrobial resistance or virulence. Previous transcriptomic studies done under comparable conditions had not been able to highlight these transcriptional modifications. These results highlight the importance of validating RGs prior to the normalization of transcriptional expression levels of targeted genes. This accurate methodology can be extended to any gene of interest in Y. pestis. More generally, the same workflow can be applied to identify and validate appropriate RGs in other bacteria to study transcriptional variations. Nature Publishing Group UK 2019-02-21 /pmc/articles/PMC6385181/ /pubmed/30792499 http://dx.doi.org/10.1038/s41598-019-39072-x Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Koch, Lionel Poyot, Thomas Schnetterle, Marine Guillier, Sophie Soulé, Estelle Nolent, Flora Gorgé, Olivier Neulat-Ripoll, Fabienne Valade, Eric Sebbane, Florent Biot, Fabrice Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions |
title | Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions |
title_full | Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions |
title_fullStr | Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions |
title_full_unstemmed | Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions |
title_short | Transcriptomic studies and assessment of Yersinia pestis reference genes in various conditions |
title_sort | transcriptomic studies and assessment of yersinia pestis reference genes in various conditions |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385181/ https://www.ncbi.nlm.nih.gov/pubmed/30792499 http://dx.doi.org/10.1038/s41598-019-39072-x |
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