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

Detalhes bibliográficos
Autor(a) principal: Oliveira, Marina Martins de
Data de Publicação: 2020
Outros Autores: Livramento, Kalynka Gabriella do, Magalhães, Maísa Lamounier, Paiva, Luciano Vilela, Peconick, Ana Paula
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Research, Society and Development
Texto Completo: https://rsdjournal.org/index.php/rsd/article/view/9702
Resumo: 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.
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spelling Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetesValidación de los genes de referencia para RT-qPCR en tejido cardíaco de ratón inducido por obesidad y diabetesValidação dos genes de referência para RT-qPCR em tecido cardíaco de ratos induzidos à obesidade e ao diabetesCorazónRodendNormalizaciónRefFindeGAPDH.CoraçãoRoedorNormalizaçãoRefFindeGAPDH.HeartRodendNormalizationRefFindeGAPDH.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.La validación de genes de referencia es un paso fundamental para análisis de RT-qPCR. El presente trabajo tuvo como objetivo identificar y validar genes de referencia para RT-qPCR en tejido cardíaco de ratas de la especie Rattus norvegicus albinus, sometidas a obesidad asociada o no a diabetes mellitus tipo 2. Fueran introducidos los cambios metabólicos a los 42 días de vida y se realizó la eutanasia a los 70 días. Los ápices del corazón fueran destinados a la extracción de RNA. La técnica RT-qPCR se realizó en termociclador propio, la eficiencia de los cebadores encontrados por el software LinReg y la estabilidad de la expresión de los genes de referencia en las muestras se analizó mediante el algoritmo RefFinder. Los candidatos a genes de referencia fueron GAPDH, POLR2A, RPL32 y RPL4, y el gen diana utilizado para verificar las diferencias en la expresión génica de los candidatos a genes de referencia fue CMA1. Los animales obesos mostraron una disminución en la expresión del gen CMA1 en comparación con los dos genes de referencia más estables. Lo contrario ocurre cuando se compara con los dos genes de referencia menos estables. Los genes GAPDH y POLR2A son los mejores para normalizar las reacciones con las muestras en cuestión. No existe un gen de referencia universal, lo que requiere una validación sistemática para cada situación. El uso de genes de referencia no validados puede comprometer la interpretación de la expresión de los genes diana, lo que evitaría el reflejo de la situación real.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.Research, Society and Development2020-11-08info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://rsdjournal.org/index.php/rsd/article/view/970210.33448/rsd-v9i11.9702Research, Society and Development; Vol. 9 No. 11; e1599119702Research, Society and Development; Vol. 9 Núm. 11; e1599119702Research, Society and Development; v. 9 n. 11; e15991197022525-3409reponame:Research, Society and Developmentinstname:Universidade Federal de Itajubá (UNIFEI)instacron:UNIFEIenghttps://rsdjournal.org/index.php/rsd/article/view/9702/8637Copyright (c) 2020 Marina Martins de Oliveira; Kalynka Gabriella do Livramento; Maísa Lamounier Magalhães; Luciano Vilela Paiva; Ana Paula Peconickhttps://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessOliveira, Marina Martins deLivramento, Kalynka Gabriella doMagalhães, Maísa LamounierPaiva, Luciano VilelaPeconick, Ana Paula2020-12-10T23:37:57Zoai:ojs.pkp.sfu.ca:article/9702Revistahttps://rsdjournal.org/index.php/rsd/indexPUBhttps://rsdjournal.org/index.php/rsd/oairsd.articles@gmail.com2525-34092525-3409opendoar:2024-01-17T09:31:55.144238Research, Society and Development - Universidade Federal de Itajubá (UNIFEI)false
dc.title.none.fl_str_mv Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes
Validación de los genes de referencia para RT-qPCR en tejido cardíaco de ratón inducido por obesidad y diabetes
Validação dos genes de referência para RT-qPCR em tecido cardíaco de ratos induzidos à obesidade e ao diabetes
title Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes
spellingShingle Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes
Oliveira, Marina Martins de
Corazón
Rodend
Normalización
RefFinde
GAPDH.
Coração
Roedor
Normalização
RefFinde
GAPDH.
Heart
Rodend
Normalization
RefFinde
GAPDH.
title_short Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes
title_full Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes
title_fullStr Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes
title_full_unstemmed Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes
title_sort Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes
author Oliveira, Marina Martins de
author_facet Oliveira, Marina Martins de
Livramento, Kalynka Gabriella do
Magalhães, Maísa Lamounier
Paiva, Luciano Vilela
Peconick, Ana Paula
author_role author
author2 Livramento, Kalynka Gabriella do
Magalhães, Maísa Lamounier
Paiva, Luciano Vilela
Peconick, Ana Paula
author2_role author
author
author
author
dc.contributor.author.fl_str_mv Oliveira, Marina Martins de
Livramento, Kalynka Gabriella do
Magalhães, Maísa Lamounier
Paiva, Luciano Vilela
Peconick, Ana Paula
dc.subject.por.fl_str_mv Corazón
Rodend
Normalización
RefFinde
GAPDH.
Coração
Roedor
Normalização
RefFinde
GAPDH.
Heart
Rodend
Normalization
RefFinde
GAPDH.
topic Corazón
Rodend
Normalización
RefFinde
GAPDH.
Coração
Roedor
Normalização
RefFinde
GAPDH.
Heart
Rodend
Normalization
RefFinde
GAPDH.
description 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.
publishDate 2020
dc.date.none.fl_str_mv 2020-11-08
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://rsdjournal.org/index.php/rsd/article/view/9702
10.33448/rsd-v9i11.9702
url https://rsdjournal.org/index.php/rsd/article/view/9702
identifier_str_mv 10.33448/rsd-v9i11.9702
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv https://rsdjournal.org/index.php/rsd/article/view/9702/8637
dc.rights.driver.fl_str_mv https://creativecommons.org/licenses/by/4.0
info:eu-repo/semantics/openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/4.0
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Research, Society and Development
publisher.none.fl_str_mv Research, Society and Development
dc.source.none.fl_str_mv Research, Society and Development; Vol. 9 No. 11; e1599119702
Research, Society and Development; Vol. 9 Núm. 11; e1599119702
Research, Society and Development; v. 9 n. 11; e1599119702
2525-3409
reponame:Research, Society and Development
instname:Universidade Federal de Itajubá (UNIFEI)
instacron:UNIFEI
instname_str Universidade Federal de Itajubá (UNIFEI)
instacron_str UNIFEI
institution UNIFEI
reponame_str Research, Society and Development
collection Research, Society and Development
repository.name.fl_str_mv Research, Society and Development - Universidade Federal de Itajubá (UNIFEI)
repository.mail.fl_str_mv rsd.articles@gmail.com
_version_ 1797052831586320384

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