Avaliação de genes de referência em diferentes tecidos de bovinos de corte

Abstract

The objective of the present study was to evaluate the stability of reference candidate genes Cancer Susceptibility Candidate 3 (CASC3), Eukaryotic Translation Elongation Factor 1 Alpha 2 (EEF1A2), Hydroxymethylbilane Synthase (HMBS), 18S Ribosomal RNA (18S), Actin Beta ), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and Ubiquitin C (UBC), and to select the most stable genes in the tissues: liver, muscle and jejunum of purebred (Nellore) or crossbred (Nellore x Angus) steers under different diets to be used in RT-qPCR analyses. In addition, to validate the selection of candidate reference genes, expression of the target genes Maltase-Glucoamylase 2 (MGAM), Solute Carrier Family 2 Member 1 (SLC2A1), Stearoyl-CoA Desaturase (SCD), Acyl-CoA Oxidase 1 ( ACOX1) and Fatty Acid Synthase (FAS) were evaluated using the different genes. Fourteen Nellore steers and fourteen crossbred steers were used, with half of the animals in each genetic group were fed a diet containing whole shelled corn (GMI) and the other half whole shelled corn and sugarcane bagasse (GMIB). Stability analyses was performed through the online program RefFinder, which combines the algorithms GeNorm, NormFinder, Bestkeeper and Delta-Cq. The ACTB was among the three most stable genes for each tissue evaluated, and for the other genes the stability and inclusion of the genes have changed depending on tissue and breed. For the muscle, the most stable genes were 18S, ACTB and CASC3; for the liver, HMBS, ACTB and 18S; and for the jejunum GAPDH, ACTB and CASC3, regardless of breed and diet. The use of the most stable and less stable genes as reference for normalization of the target genes FAS, ACOX1, MGAM and SCD may change RT-qPCR results causing errors in the analyses of the data. The use of more stable and less stable reference gene sets may lead to different conclusions about the expression profile of the target gene studied. The results found in the validation of the reference genes reinforce the fact that the selection of the most suitable reference genes for each experiment is of paramount importance to ensure the reliability of the gene expression studies so that they can be applied in practice. Thus, the study demonstrates the importance of making previous analyzes for each experimental condition to be studied.