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Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells

BACKGROUND: Selecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation i...

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Autores principales: Vossaert, Liesbeth, O’Leary, Thomas, Van Neste, Christophe, Heindryckx, Björn, Vandesompele, Jo, De Sutter, Petra, Deforce, Dieter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848990/
https://www.ncbi.nlm.nih.gov/pubmed/24028740
http://dx.doi.org/10.1186/1471-2199-14-21
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author Vossaert, Liesbeth
O’Leary, Thomas
Van Neste, Christophe
Heindryckx, Björn
Vandesompele, Jo
De Sutter, Petra
Deforce, Dieter
author_facet Vossaert, Liesbeth
O’Leary, Thomas
Van Neste, Christophe
Heindryckx, Björn
Vandesompele, Jo
De Sutter, Petra
Deforce, Dieter
author_sort Vossaert, Liesbeth
collection PubMed
description BACKGROUND: Selecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation itself introduces changes whereby reference gene stability may be influenced. RESULTS: In this study, we evaluated the stability of various references during retinoic acid-induced (2 microM) differentiation of hES cells. Out of 12 candidate references, beta-2-microglobulin, ribosomal protein L13A and Alu repeats are found to be the most stable for this experimental set-up. CONCLUSIONS: Our results show that some of the commonly used reference genes are actually not amongst the most stable loci during hES cell differentiation promoted by retinoic acid. Moreover, a novel normalization strategy based on expressed Alu repeats is validated for use in hES cell experiments.
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spelling pubmed-38489902013-12-04 Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells Vossaert, Liesbeth O’Leary, Thomas Van Neste, Christophe Heindryckx, Björn Vandesompele, Jo De Sutter, Petra Deforce, Dieter BMC Mol Biol Research Article BACKGROUND: Selecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation itself introduces changes whereby reference gene stability may be influenced. RESULTS: In this study, we evaluated the stability of various references during retinoic acid-induced (2 microM) differentiation of hES cells. Out of 12 candidate references, beta-2-microglobulin, ribosomal protein L13A and Alu repeats are found to be the most stable for this experimental set-up. CONCLUSIONS: Our results show that some of the commonly used reference genes are actually not amongst the most stable loci during hES cell differentiation promoted by retinoic acid. Moreover, a novel normalization strategy based on expressed Alu repeats is validated for use in hES cell experiments. BioMed Central 2013-09-12 /pmc/articles/PMC3848990/ /pubmed/24028740 http://dx.doi.org/10.1186/1471-2199-14-21 Text en Copyright © 2013 Vossaert et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Vossaert, Liesbeth
O’Leary, Thomas
Van Neste, Christophe
Heindryckx, Björn
Vandesompele, Jo
De Sutter, Petra
Deforce, Dieter
Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells
title Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells
title_full Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells
title_fullStr Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells
title_full_unstemmed Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells
title_short Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells
title_sort reference loci for rt-qpcr analysis of differentiating human embryonic stem cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848990/
https://www.ncbi.nlm.nih.gov/pubmed/24028740
http://dx.doi.org/10.1186/1471-2199-14-21
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