Estudo de genes de referência em Zebrafish (Danio rerio) transgênicos superexpressando o hormônio do crescimento

Abstract

Real-time quantitative PCR is one of the most important techniques in the analysis of gene expression and needs the use of reference genes. These genes have the function of normalizing the reaction data, having an important role in the outcome and success of the analysis. Reference genes must have significant and stable expression in tissues and in all experimental conditions. Zebrafish (Danio rerio) reference genes have been studied, however, as far as we know, there are no studies for the transgenic models of this species. The gh-transgenic zebrafish is a strain called F0104, developed to overexpress the growth hormone from silverside (Odonthestes argentinensis). It is assumed that the transgenic process may influence the expression levels of commonly used reference genes, leading to the need to validate specific reference genes for gene expression studies in transgenic zebrafish. In this context, the objective of the present study was to analyze the stability of genes actb1, actb2, atp1a, b2m, eif2b, ef1α, igf1, gapdh, rpl2, rpl7, rpl13α, tuba, and 18s in transgenic and non-transgenic zebrafish, in order to evaluate their potential use as reference genes. For this, adult animals of both sexes, gh-transgenic (n = 21) and non-transgenic (n = 21), constituted the experimental groups. Liver, brain, intestine and muscle samples were collected. Every three samples of each organ collected, a pool was made, totalizing seven pools. After the steps of RNA extraction, cDNA synthesis and real-time qPCR, the analyzes were performed with software dCt, GeNorm, NormFinder, BestKeeper and RefFinder. The most stable genes for the gh- transgenic zebrafish group were rpl13α, ef1α, rpl7 and rpl2, whereas the less stable genes were the gapdh, 18s and tuba genes, respectively. From the obtained data, it was verified that the tissue that most influenced in the instability of the genes were the intestine and the liver.This study also showed that even genes considered to be constitutive and commonly used as normalizers in studies with non-transgenic zebrafish, such as gapdh, 18s and tuba, suffered variations between different tissues of both transgenic and non-transgenic animals. Finally, it is concluded that the genes rpl13α, ef1α, rpl7 and rpl2 have high stability, being possible to be used as reference genes in studies involving analysis of gene expression in zebrafish (Dano rerio) and that genes such as gapdh, 18s and tuba have low stability and therefore are not suitable as reference genes for the studied groups and tissues.