Please use this identifier to cite or link to this item: http://www.arca.fiocruz.br/handle/icict/18597
Title: Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions
Authors: Mafra, Valéria
Kubo, Karen S.
Ferreira, Marcio Alves
Alves, Marcelo Ribeiro
Stuart, Rodrigo M.
Boava, Leonardo P.
Rodrigues, Carolina M.
Machado, Marcos A.
Affilliation: Centro de Citricultura Sylvio Moreira. Laboratório de Biotecnologia. Cordeirópolis, SP, Brasil / Universidade Estadual de Campinas. Instituto de Biologia. Campinas, SP, Brasil.
Centro de Citricultura Sylvio Moreira. Laboratório de Biotecnologia. Cordeirópolis, SP, Brasil / Universidade Estadual de Campinas. Instituto de Biologia. Campinas, SP, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Biologia. Departamento de Genética. Laboratório de Genética Molecular Vegetal. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil.
Centro de Citricultura Sylvio Moreira. Laboratório de Biotecnologia. Cordeirópolis, SP, Brasil / Universidade Estadual de Campinas. Instituto de Biologia. Campinas, SP, Brasil.
Centro de Citricultura Sylvio Moreira. Laboratório de Biotecnologia. Cordeirópolis, SP, Brasil.
Centro de Citricultura Sylvio Moreira. Laboratório de Biotecnologia. Cordeirópolis, SP, Brasil.
Centro de Citricultura Sylvio Moreira. Laboratório de Biotecnologia. Cordeirópolis, SP, Brasil.
Abstract: 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.
Keywords: Reference Genes
Citrus Genotypes
Different Experimental Conditions
Accurate Transcript Normalization
keywords: Genótipos de citros
Diferentes Condições Experimentais
Genes de referência
Normalização Exata de Transcrição
Issue Date: 2012
Publisher: Public Library of Science
Citation: MAFRA, Valéria; et al. Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions. Plos One, v.7, n.2, e31263, 11p, Feb. 2012.
DOI: 10.1371/journal.pone.0031263
ISSN: 1932-6203
Copyright: open access
Appears in Collections:IOC - Artigos de Periódicos

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