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

Autores

DOI:

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

Palavras-chave:

Coração; Roedor; Normalização; RefFinde; GAPDH.

Resumo

A validação dos genes de referência é uma etapa essencial para qualquer análise de RT-qPCR. Dessa forma, o presente trabalho teve como objetivo identificar e validar genes de referência para RT-qPCR em tecido cardíaco de ratos da espécie Rattus norvegicus albinus, submetidos à obesidade associada ou não ao diabetes mellitus tipo 2. Para isso, as alterações metabólicas foram induzidas no 42º dia de vida e a eutanásia foi realizada no 70º dia. Os ápices cardíacos foram coletados e destinam-se à extração de RNA. A técnica de RT-qPCR foi realizada em termociclador próprio, a eficiência dos primers encontrados pelo software LinReg e a estabilidade da expressão dos genes de referência nas amostras foram analisadas pelo algorítmo RefFinder. Os candidatos a genes de referência foram GAPDH, POLR2A, RPL32 e RPL4 e o gene alvo usado para verificar as diferenças na expressão gênica dos candidatos a genes de referência foi CMA1. Os animais obesos apresentaram uma diminuição na expressão do gene CMA1 quando comparados aos dois genes de referência mais estáveis. O oposto ocorre quando comparado aos dois genes de referência menos estáveis. Os genes GAPDH e POLR2A são os melhores para normalizar as reações com as amostras em questão. Não há gene de referência universal para todas as situações, o que requer validação sistemática para cada situação. O uso de genes de referência não validados pode comprometer a interpretação da expressão dos genes alvo, o que impediria a reflexão da situação real.

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Publicado

08/11/2020

Como Citar

OLIVEIRA, M. M. de; LIVRAMENTO, K. G. do; MAGALHÃES, M. L.; PAIVA, L. V.; PECONICK, A. P. Validação dos genes de referência para RT-qPCR em tecido cardíaco de ratos induzidos à obesidade e ao diabetes. Research, Society and Development, [S. l.], v. 9, n. 11, p. e1599119702, 2020. DOI: 10.33448/rsd-v9i11.9702. Disponível em: https://rsdjournal.org/index.php/rsd/article/view/9702. Acesso em: 18 set. 2024.

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Ciências Agrárias e Biológicas