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Selection of reference genes for qPCR in hairy root cultures of peanut

BACKGROUND: Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of the...

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Autores principales: Condori, Jose, Nopo-Olazabal, Cesar, Medrano, Giuliana, Medina-Bolivar, Fabricio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199266/
https://www.ncbi.nlm.nih.gov/pubmed/21985172
http://dx.doi.org/10.1186/1756-0500-4-392
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author Condori, Jose
Nopo-Olazabal, Cesar
Medrano, Giuliana
Medina-Bolivar, Fabricio
author_facet Condori, Jose
Nopo-Olazabal, Cesar
Medrano, Giuliana
Medina-Bolivar, Fabricio
author_sort Condori, Jose
collection PubMed
description BACKGROUND: Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. RESULTS: A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc). CONCLUSIONS: This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.
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spelling pubmed-31992662011-10-24 Selection of reference genes for qPCR in hairy root cultures of peanut Condori, Jose Nopo-Olazabal, Cesar Medrano, Giuliana Medina-Bolivar, Fabricio BMC Res Notes Research Article BACKGROUND: Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. RESULTS: A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc). CONCLUSIONS: This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots. BioMed Central 2011-10-10 /pmc/articles/PMC3199266/ /pubmed/21985172 http://dx.doi.org/10.1186/1756-0500-4-392 Text en Copyright ©2011 Medina-Bolivar 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
Condori, Jose
Nopo-Olazabal, Cesar
Medrano, Giuliana
Medina-Bolivar, Fabricio
Selection of reference genes for qPCR in hairy root cultures of peanut
title Selection of reference genes for qPCR in hairy root cultures of peanut
title_full Selection of reference genes for qPCR in hairy root cultures of peanut
title_fullStr Selection of reference genes for qPCR in hairy root cultures of peanut
title_full_unstemmed Selection of reference genes for qPCR in hairy root cultures of peanut
title_short Selection of reference genes for qPCR in hairy root cultures of peanut
title_sort selection of reference genes for qpcr in hairy root cultures of peanut
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199266/
https://www.ncbi.nlm.nih.gov/pubmed/21985172
http://dx.doi.org/10.1186/1756-0500-4-392
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