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Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process

Induced pluripotent stem cell (iPS) reprogramming allows to turn a differentiated somatic cell into a pluripotent cell. This process is accompanied by many changes in fundamental cell properties, such as energy production, cell-to-cell interactions, cytoskeletal organization, and others. Real-time q...

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Autores principales: Panina, Yulia, Germond, Arno, Masui, Shinji, Watanabe, Tomonobu M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992140/
https://www.ncbi.nlm.nih.gov/pubmed/29880849
http://dx.doi.org/10.1038/s41598-018-26707-8
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author Panina, Yulia
Germond, Arno
Masui, Shinji
Watanabe, Tomonobu M.
author_facet Panina, Yulia
Germond, Arno
Masui, Shinji
Watanabe, Tomonobu M.
author_sort Panina, Yulia
collection PubMed
description Induced pluripotent stem cell (iPS) reprogramming allows to turn a differentiated somatic cell into a pluripotent cell. This process is accompanied by many changes in fundamental cell properties, such as energy production, cell-to-cell interactions, cytoskeletal organization, and others. Real-time quantitative polymerase chain reaction (RT-qPCR) can be used as a quantitative method of gene expression analysis to investigate iPS reprogramming but it requires a validation of reference genes for the accurate assessment of target genes’ expression. Currently, studies evaluating the performance of reference genes during iPS reprogramming are lacking. In this study we analysed the stability of 12 housekeeping genes during 20 days of iPS reprogramming of murine cells based on statistical analyses of RT-qPCR data using five different statistical algorithms. This study reports strong variations in housekeeping gene stability during the reprogramming process. Most stable genes were Atp5f1, Pgk1 and Gapdh, while the least stable genes were Rps18, Hprt, Tbp and Actb. The results were validated by a proof-of-point qPCR experiment with pluripotent markers Nanog, Rex1 and Oct4 normalized to the best and the worst reference gene identified by the analyses. Overall, this study and its implications are particularly relevant to investigations on the cell-state and pluripotency in iPS reprogramming.
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spelling pubmed-59921402018-06-21 Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process Panina, Yulia Germond, Arno Masui, Shinji Watanabe, Tomonobu M. Sci Rep Article Induced pluripotent stem cell (iPS) reprogramming allows to turn a differentiated somatic cell into a pluripotent cell. This process is accompanied by many changes in fundamental cell properties, such as energy production, cell-to-cell interactions, cytoskeletal organization, and others. Real-time quantitative polymerase chain reaction (RT-qPCR) can be used as a quantitative method of gene expression analysis to investigate iPS reprogramming but it requires a validation of reference genes for the accurate assessment of target genes’ expression. Currently, studies evaluating the performance of reference genes during iPS reprogramming are lacking. In this study we analysed the stability of 12 housekeeping genes during 20 days of iPS reprogramming of murine cells based on statistical analyses of RT-qPCR data using five different statistical algorithms. This study reports strong variations in housekeeping gene stability during the reprogramming process. Most stable genes were Atp5f1, Pgk1 and Gapdh, while the least stable genes were Rps18, Hprt, Tbp and Actb. The results were validated by a proof-of-point qPCR experiment with pluripotent markers Nanog, Rex1 and Oct4 normalized to the best and the worst reference gene identified by the analyses. Overall, this study and its implications are particularly relevant to investigations on the cell-state and pluripotency in iPS reprogramming. Nature Publishing Group UK 2018-06-07 /pmc/articles/PMC5992140/ /pubmed/29880849 http://dx.doi.org/10.1038/s41598-018-26707-8 Text en © The Author(s) 2018 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
Panina, Yulia
Germond, Arno
Masui, Shinji
Watanabe, Tomonobu M.
Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process
title Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process
title_full Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process
title_fullStr Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process
title_full_unstemmed Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process
title_short Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process
title_sort validation of common housekeeping genes as reference for qpcr gene expression analysis during ips reprogramming process
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992140/
https://www.ncbi.nlm.nih.gov/pubmed/29880849
http://dx.doi.org/10.1038/s41598-018-26707-8
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