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Evaluation of reference genes for reverse transcription-quantitative PCR assays in organs of zebrafish exposed to glyphosate-based herbicide, Roundup

Published online by Cambridge University Press:  27 November 2017

M. L. Jaramillo ,
A. G. Pereira ,
C. E. Davico ,
L. Nezzi ,
D. Ammar ,
Y. M. R. Müller  and
E. M. Nazari
M. L. Jaramillo
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
A. G. Pereira
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
C. E. Davico
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
L. Nezzi
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
D. Ammar
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil Centro Universitário – Católica de Santa Catarina, 89203-005 Joinville, Santa Catarina, Brazil
Y. M. R. Müller
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
E. M. Nazari*
Affiliation:
Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil
*
†E-mail: evelise.nazari@ufsc.br
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Abstract

Roundup is a glyphosate-based herbicide (GBH) widely used in agriculture and may cause toxic effects in non-target organisms. Model organisms, as zebrafish, and analysis of gene expression by reverse transcription-quantitative PCR (RT-qPCR) could be used to better understand the Roundup toxicity. A prerequisite for RT-qPCR is the availability of appropriate reference genes; however, they have not been described for Roundup-exposed fish. The aim of this study was to evaluate the expression stability of six reference genes (rpl8, β-act, gapdh, b2m, ef1α, hprt1) and one expressed repetitive element (hatn10) in organs of males (brain, gill, testis) and females (ovary) of zebrafish exposed to Roundup WG at three concentrations (0.065, 0.65 and 6.5 mg N-(phosphonomethyl) glycine/l) for 7 days. Genes were ranked by geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder, and their best combinations were determined by geNorm and NormFinder programs. The two most stable ranked genes were specific to each organ: gill (β-act; rpl8); brain (rpl8; β-act); testis (ef1α; gapdh); and ovary (rpl8; hprt1). The cat transcript level was used to evaluate the effect of normalization with these reference genes. These are the first suitable reference genes described for the analysis of gene expression in organs of Roundup-exposed zebrafish, and will allow investigations of the molecular mechanisms of Roundup toxicity.

Keywords

Roundup WGnormalizationexpression stabilityreverse transcription-quantitative PCRDanio rerio

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Type
Research Article
Information
animal , Volume 12 , Issue 7 , July 2018 , pp. 1424 - 1434
Copyright
© The Animal Consortium 2017 

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