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Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii

qRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all exper...

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Autores principales: Jiang, Hucheng, Qian, Zhaojun, Lu, Wei, Ding, Huaiyu, Yu, Hongwei, Wang, Hui, Li, Jiale
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613269/
https://www.ncbi.nlm.nih.gov/pubmed/26370979
http://dx.doi.org/10.3390/ijms160921591
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author Jiang, Hucheng
Qian, Zhaojun
Lu, Wei
Ding, Huaiyu
Yu, Hongwei
Wang, Hui
Li, Jiale
author_facet Jiang, Hucheng
Qian, Zhaojun
Lu, Wei
Ding, Huaiyu
Yu, Hongwei
Wang, Hui
Li, Jiale
author_sort Jiang, Hucheng
collection PubMed
description qRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all experimental conditions, and research on suitable reference genes in red swamp crawfish (Procambarus clarkii) is particularly scarce. In this study, eight commonly used crustacean reference genes were chosen from P. clarkii transcriptome data and investigated as potential candidates for normalization of qRT-PCR data. Expression of these genes under different experimental conditions was examined by qRT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, geNorm, NormFinder and BestKeeper. A final comprehensive ranking determined that EIF and 18S were the optimal reference genes for expression data from different tissues, while TBP and EIF were optimal for expression data from different ovarian developmental stages. To our knowledge, this is the first systematic analysis of reference genes for normalization of qRT-PCR data in P. clarkii. These results will facilitate more accurate and reliable expression studies of this and other crustacean species.
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spelling pubmed-46132692015-10-26 Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii Jiang, Hucheng Qian, Zhaojun Lu, Wei Ding, Huaiyu Yu, Hongwei Wang, Hui Li, Jiale Int J Mol Sci Article qRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all experimental conditions, and research on suitable reference genes in red swamp crawfish (Procambarus clarkii) is particularly scarce. In this study, eight commonly used crustacean reference genes were chosen from P. clarkii transcriptome data and investigated as potential candidates for normalization of qRT-PCR data. Expression of these genes under different experimental conditions was examined by qRT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, geNorm, NormFinder and BestKeeper. A final comprehensive ranking determined that EIF and 18S were the optimal reference genes for expression data from different tissues, while TBP and EIF were optimal for expression data from different ovarian developmental stages. To our knowledge, this is the first systematic analysis of reference genes for normalization of qRT-PCR data in P. clarkii. These results will facilitate more accurate and reliable expression studies of this and other crustacean species. MDPI 2015-09-08 /pmc/articles/PMC4613269/ /pubmed/26370979 http://dx.doi.org/10.3390/ijms160921591 Text en © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Jiang, Hucheng
Qian, Zhaojun
Lu, Wei
Ding, Huaiyu
Yu, Hongwei
Wang, Hui
Li, Jiale
Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii
title Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii
title_full Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii
title_fullStr Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii
title_full_unstemmed Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii
title_short Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii
title_sort identification and characterization of reference genes for normalizing expression data from red swamp crawfish procambarus clarkii
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613269/
https://www.ncbi.nlm.nih.gov/pubmed/26370979
http://dx.doi.org/10.3390/ijms160921591
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