Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes

Marina Martins De Oliveira,Maísa Lamounier Magalhães,Kalynka Gabriella Do Livramento,Ana Paula Peconick,Luciano Vilela Paiva

doi:10.33448/rsd-v9i11.9702

Marina Martins De Oliveira, Maísa Lamounier Magalhães + Show 3 more

Open Access

https://doi.org/10.33448/rsd-v9i11.9702

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Abstract

The validation of reference genes is an essential step for any RT-qPCR analysis. In this way, the present paper aimed to identify and validate reference genes for RT-qPCR in cardiac tissue of rats of the Rattus norvegicus albinus specie, submitted to obesity associated or not to type 2 diabetes mellitus. For this, the metabolic changes were induced at the 42nd day of life and the euthanasia was performed on the 70th day. The heart apexes were collected and destinates for RNA extraction. The RT-qPCR technique was performed in own thermocycler, the efficiency of the primers found by the LinReg software and the stability of the expression of the reference genes in the samples was analyzed by the RefFinder algorithm. The candidates for reference genes were GAPDH, POLR2A, RPL32, and RPL4 and the target gene used to verify the differences in gene expression of candidates for reference genes was CMA1. The 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. The GAPDH and POLR2A genes are the best to normalize the reactions with the samples in question. There is no universal reference gene for all situations, which requires systematic validation for each situation. The use of unvalidated reference genes may compromise the interpretation of the expression of the target genes, which would prevent the reflection of the actual situation.

Highlights

The obesity and diabetes are multifactorial diseases that can cause damage to other organic systems, such as the renal and cardiac (Cefalu et al, 2015)
The E values ranged from 81% to 95% and R2 values were higher than 0.97, indicating that the cDNA model was successfully duplicated at the end of each cycle. 3.2 Expression and stability of the reference genes candidates The expression of the reference genes candidates are shown in Figure 1, with the variation, expressed in boxplot diagrams
The glyceraldehyde-6phosphate dehydrogenase (GAPDH) gene is commonly used as a reference gene for RT-qPCR in murine models, its stability varies (Eissa et al, 2017)

Summary

Introduction

The obesity and diabetes are multifactorial diseases that can cause damage to other organic systems, such as the renal and cardiac (Cefalu et al, 2015). The hyperglycemia, resulting from the deficiency of insulin production or the resistance to it, generates the formation of reactive oxygen species (ROS), and the chronic exposure to ROS may increase cardiomyocyte apoptosis and accumulation of fibrous tissue in the heart (Shuckla et al, 2017). In this context, there is an enzyme named chymase, that may be involved in this heart condition, considering that it participates in 80% of cardiac angiotensin II synthesis (Ahmad et al, 2014).

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