Abstract
Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes.
Highlights
Northern-blotting, ribonuclease protection assay, microarray, RNA-Seq, semi-quantitative RT-PCR, and quantitative RT-PCR are among the most currently used techniques for evaluation of gene expression levels
It was used individual methods to obtain independent rankings of gene expression stability of candidate reference gene (RG) in O. humensis and an algorithm that deduces a consensus ranking among all methods (RefFinder) such as presented in recent studies about RGs (Taki et al, 2014; Xu et al, 2014; Huang et al, 2015)
The dCt method compares the relative expression of gene pairs with their standard deviation (SD) values to get the most stable
Summary
Northern-blotting, ribonuclease protection assay, microarray, RNA-Seq, semi-quantitative RT-PCR, and quantitative RT-PCR (qRT-PCR) are among the most currently used techniques for evaluation of gene expression levels. The latter stands out for fast readout, high throughput and high automation potential (Tang et al, 2012). The relative quantification describes the change in the expression pattern of genes of interest when subjected to certain controlled situations compared to the constant expression of reference genes (RGs), known as housekeeping genes under same conditions. It is extremely important to have a stable and constant expression pattern of RGs regardless of gender, age, organ or tissue, and even after environmental changes. It is necessary to validate diverse RGs for various species and their stages of development in different tissues, as well as for each type of controlled environmental condition (Livak and Schmittgen, 2001; Zheng and Sun, 2011)
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