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Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood
The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Molecular Diversity Preservation International (MDPI)
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3233434/ https://www.ncbi.nlm.nih.gov/pubmed/22174628 http://dx.doi.org/10.3390/ijms12117732 |
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author | Peletto, Simone Bertuzzi, Simone Campanella, Chiara Modesto, Paola Maniaci, Maria Grazia Bellino, Claudio Ariello, Dario Quasso, Antonio Caramelli, Maria Acutis, Pier Luigi |
author_facet | Peletto, Simone Bertuzzi, Simone Campanella, Chiara Modesto, Paola Maniaci, Maria Grazia Bellino, Claudio Ariello, Dario Quasso, Antonio Caramelli, Maria Acutis, Pier Luigi |
author_sort | Peletto, Simone |
collection | PubMed |
description | The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells. |
format | Online Article Text |
id | pubmed-3233434 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Molecular Diversity Preservation International (MDPI) |
record_format | MEDLINE/PubMed |
spelling | pubmed-32334342011-12-15 Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood Peletto, Simone Bertuzzi, Simone Campanella, Chiara Modesto, Paola Maniaci, Maria Grazia Bellino, Claudio Ariello, Dario Quasso, Antonio Caramelli, Maria Acutis, Pier Luigi Int J Mol Sci Article The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells. Molecular Diversity Preservation International (MDPI) 2011-11-09 /pmc/articles/PMC3233434/ /pubmed/22174628 http://dx.doi.org/10.3390/ijms12117732 Text en © 2011 by the authors; licensee MDPI, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0 This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Peletto, Simone Bertuzzi, Simone Campanella, Chiara Modesto, Paola Maniaci, Maria Grazia Bellino, Claudio Ariello, Dario Quasso, Antonio Caramelli, Maria Acutis, Pier Luigi Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood |
title | Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood |
title_full | Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood |
title_fullStr | Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood |
title_full_unstemmed | Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood |
title_short | Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood |
title_sort | evaluation of internal reference genes for quantitative expression analysis by real-time pcr in ovine whole blood |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3233434/ https://www.ncbi.nlm.nih.gov/pubmed/22174628 http://dx.doi.org/10.3390/ijms12117732 |
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