Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions

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 ( 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 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 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 , and . 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 to be the most stable gene expression when only the treatments were considered. On the other hand, was the least stable gene in the control and was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that is the most stable gene whereas and are the least stable genes, even being considered two constitutive genes.