Validação de genes de referência para RT-qPCR em tecido cardíaco de ratos submetidos à obesidade e ao diabetes

Abstract

The present work aims to identify and validate reference genes for RT-qPCR in cardiac tissue of rats of the Rattusnorvegicusalbinus species, submitted to obesity associated or not to type 2 diabetes mellitus. For this, the metabolic changes were induced at 42 days of life, with euthanasia being performed on the 70th day. The hearts of the animals were collected and the cardiac apex was assigned to the molecular techniques for RNA extraction, performed with TRIzol®. Candidates for reference genes were GAPDH, POLR2A, RPL32 and RPL4. The RT-qPCR technique was done in thermocycler, with the efficiency of the primers found by the LinReg software and the stability of the expression of the reference genes in the samples analyzed by the RefFinder algorithm. The target gene used to verify the difference in gene expression of candidates for reference genes was CMA1. The results suggest that obese animals showed a decrease in CMA1 gene expression when compared to the two most stable reference genes. The opposite occurs when it is compared to the two less stable reference genes. There is no universal reference gene for all situations, which requires systematic validation for each. The use of unvalidated reference genes may compromise the expression of the target genes, which would prevent the reflection of the actual situation. The GAPDH and POLR2A genes are best at normalizing the reactions with the samples under the conditions of the present experiment.