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Looking for reference genes for real-time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae)

Authors

  • D. Majerowicz,

    Corresponding author
    1. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • M. Alves-Bezerra,

    1. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • R. Logullo,

    1. Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Brazil
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  • A. L. Fonseca-de-Souza,

    1. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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    • Present address: Coordenação de Tecnologia em Produção de Fármacos e Farmácia, Centro Universitário Estadual da Zona Oeste, Brazil

  • J. R. Meyer-Fernandes,

    1. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
    2. Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Brazil
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  • G. R. C. Braz,

    1. Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Brazil
    2. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil
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  • K. C. Gondim

    Corresponding author
    1. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
    2. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil
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  • This work is dedicated to Dr Martha Meriwether Sorenson who has reviewed with outstanding scientific criticism hundreds of manuscripts produced by the researchers of Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brazil, in the last 27 years

David Majerowicz and Katia C. Gondim, Av. Carlos Chagas Filho, 373, Ilha do Fundão, Rio de Janeiro, RJ, 21941-902, Brazil. Tel.: + 55 21 2562 6785; fax: + 55 21 2270 8647; e-mail: majerowicz@bioqmed.ufrj.br (D. Majerowicz) and katia@bioqmed.ufrj.br (K. C. Gondim)

Abstract

Quantitative real-time PCR (qPCR) has become one of the most used techniques to measure gene expression. However, normalization of gene expression data against reference genes is essential, although these are usually used without any kind of validation. The expression of seven genes was compared in organs of Rhodnius prolixus under diverse conditions, using published software to test gene expression stability. Rp18S and elongation factor 1 (RpEF -1) were the most reliable genes for normalization in qPCR when gene expression in different organs was compared. Moreover, both genes were found to be the best references when transcript levels were compared in the posterior midgut of insects infected with Trypanosoma cruzi. Rp18S was also the best reference gene in the fat bodies of unfed and fed insects. By contrast, RpEF-1 was found to be the best reference gene for comparison between posterior midguts, and RpMIP or RpActin should be used to compare gene expression in the ovaries. Although Rp18S is indicated here as the best reference in most cases, reports from the literature show that it is difficult to find an optimum reference gene. Nevertheless, validation of candidate genes to be taken as references is important when new experimental conditions are tested to avoid incorrect data interpretation.

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