Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was...

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Autores principales:	Valéria Mafra, Karen S Kubo, Marcio Alves-Ferreira, Marcelo Ribeiro-Alves, Rodrigo M Stuart, Leonardo P Boava, Carolina M Rodrigues, Marcos A Machado
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/aca53c6edd67400d850ab3ff54544756
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spelling	oai:doaj.org-article:aca53c6edd67400d850ab3ff545447562021-11-18T07:28:35ZReference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.1932-620310.1371/journal.pone.0031263https://doaj.org/article/aca53c6edd67400d850ab3ff545447562012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22347455/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.Valéria MafraKaren S KuboMarcio Alves-FerreiraMarcelo Ribeiro-AlvesRodrigo M StuartLeonardo P BoavaCarolina M RodriguesMarcos A MachadoPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 2, p e31263 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Valéria Mafra Karen S Kubo Marcio Alves-Ferreira Marcelo Ribeiro-Alves Rodrigo M Stuart Leonardo P Boava Carolina M Rodrigues Marcos A Machado Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.
description	Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.
format	article
author	Valéria Mafra Karen S Kubo Marcio Alves-Ferreira Marcelo Ribeiro-Alves Rodrigo M Stuart Leonardo P Boava Carolina M Rodrigues Marcos A Machado
author_facet	Valéria Mafra Karen S Kubo Marcio Alves-Ferreira Marcelo Ribeiro-Alves Rodrigo M Stuart Leonardo P Boava Carolina M Rodrigues Marcos A Machado
author_sort	Valéria Mafra
title	Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.
title_short	Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.
title_full	Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.
title_fullStr	Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.
title_full_unstemmed	Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.
title_sort	reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/aca53c6edd67400d850ab3ff54544756
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_version_	1718423430720651264

Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.

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