Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats

Detalhes bibliográficos
Autor(a) principal: HENRIQUES, Gilberto Simeone
Data de Publicação: 2023
Outros Autores: SILVA, Adriana Gisele Hertzog da, HIRATA, Rosário Dominguez Crespo, HIRATA, Mario Hiroiuki, COZZOLINO, Sílvia Maria Franciscato
Tipo de documento: Artigo
Idioma: por
Título da fonte: Revista de Nutrição
Texto Completo: https://periodicos.puc-campinas.edu.br/nutricao/article/view/9990
Resumo: ObjectiveThe aim of the present work was to optimize the reaction conditions capable of generating variability and introducing systematic errors in the chain reaction of the polymerase used to analyze the gene expression for the testicular isoform of the angiotensin-converting enzyme.MethodsThe cDNA concentration, primer concentration, hybridization temperature and number of denaturation, hybridization and extension cycles were evaluated. For this purpose, samples of testis from Wistar rats fed a zinc containing diet were used to extract total RNA using the phenol-chloroform-isothiocyanate reaction. Stable cDNA was then generated by the reverse transcription reaction. Using specific primers, the gene of interest (testicular isoform of the angiotensin-converting enzyme) and the housekeeping gene for the expression of Glyceraldehyde-3-Phosphate Dehydrogenase were amplified. The samples were then submitted to gel eletrophoresis in agarose gel, stained with ethide bromide and visualized in a UV chamber.ResultsThe results showed that the best reaction conditions for the chain reaction by the testicular isoform polymerase of the angiotensin-converting enzyme and for Glyceraldehyde-3-Phosphate Dehydrogenase were: (1) initial cDNA concentration of 2 µg, (2) primer concentration of 200nM, (3) hybridization temperature between 57.5°C and 60.1°C and (4) 33 cycles.ConclusionIt was concluded that this optimization minimized interference of the technique, contributing to the production of true, comparative data for the testicular angiotensin- converting enzyme gene expression.
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spelling Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated ratsTranscrição reversa na determinação da expressão do mRNA para a enzima conversora de angiotensina testicular em animais tratados com zincodietangiotensin-converting enzymegene expressionreverse transcriptase polymerase chain reactionzincdietaenzima conversora de angiotensinaexpressão gênicareação em cadeia de polimerase via transcriptase reversazincoObjectiveThe aim of the present work was to optimize the reaction conditions capable of generating variability and introducing systematic errors in the chain reaction of the polymerase used to analyze the gene expression for the testicular isoform of the angiotensin-converting enzyme.MethodsThe cDNA concentration, primer concentration, hybridization temperature and number of denaturation, hybridization and extension cycles were evaluated. For this purpose, samples of testis from Wistar rats fed a zinc containing diet were used to extract total RNA using the phenol-chloroform-isothiocyanate reaction. Stable cDNA was then generated by the reverse transcription reaction. Using specific primers, the gene of interest (testicular isoform of the angiotensin-converting enzyme) and the housekeeping gene for the expression of Glyceraldehyde-3-Phosphate Dehydrogenase were amplified. The samples were then submitted to gel eletrophoresis in agarose gel, stained with ethide bromide and visualized in a UV chamber.ResultsThe results showed that the best reaction conditions for the chain reaction by the testicular isoform polymerase of the angiotensin-converting enzyme and for Glyceraldehyde-3-Phosphate Dehydrogenase were: (1) initial cDNA concentration of 2 µg, (2) primer concentration of 200nM, (3) hybridization temperature between 57.5°C and 60.1°C and (4) 33 cycles.ConclusionIt was concluded that this optimization minimized interference of the technique, contributing to the production of true, comparative data for the testicular angiotensin- converting enzyme gene expression.ObjetivoO objetivo deste trabalho foi otimizar as condições reacionais capazes de ocasionar variabilidade e de introduzir erros sistemáticos na reação em cadeia pela polimerase aplicada à análise da expressão gênica da isoforma testicular da enzima conversora de angiotensina.MétodosAvaliaram-se a concentração de cDNA, a concentração dos iniciadores, a temperatura de hibridização e o número de ciclos de desnaturação, hibridização e extensão. Para tanto, extraiu-se o RNA total por meio da reação com fenol-clorofórmio e isotiocianato de guanidina de amostras de testículos de ratos Wistar alimentados com uma ração contendo zinco. Em seguida, gerou-se o cDNA por transcrição reversa. Utilizando-se iniciadores específicos, amplificaram-se o gene de interesse (isoforma testicular da enzima conversora de angiotensina) e o gene controle Gliceraldeído-3-Fosfato-Desidrogenase. As amostras foram então aplicadas em gel de agarose e submetidas à eletroforese, coradas em brometo de etídio e visualizadas sob luz ultravioleta. ResultadosDemonstrou-se que a melhor condição reacional para a reação em cadeia pela polimerase da isoforma testicular da enzima conversora de angiotensina e do Gliceraldeído-3-Fosfato-Desidrogenase foi: (1) concentração inicial de cDNA de 2µg, (2) concentração de iniciadores de 200nM, (3) temperatura de hibridização entre 57,5°C e 60,1°C e (4) 33 ciclos.ConclusãoCom essa otimização pôde-se minimizar as interferências sobre a técnica, contribuindo-se para a obtenção de dados comparativos a respeito da expressão gênica da enzima conversora de angiotensina testicular.Núcleo de Editoração – PUC-Campinas2023-10-05info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://periodicos.puc-campinas.edu.br/nutricao/article/view/9990Brazilian Journal of Nutrition; Vol. 18 No. 6 (2005): Revista de NutriçãoRevista de Nutrição; Vol. 18 Núm. 6 (2005): Revista de NutriçãoRevista de Nutrição; v. 18 n. 6 (2005): Revista de Nutrição1678-9865reponame:Revista de Nutriçãoinstname:Pontifícia Universidade Católica de Campinas (PUC-CAMPINAS)instacron:PUC_CAMPporhttps://periodicos.puc-campinas.edu.br/nutricao/article/view/9990/7327Copyright (c) 2023 Gilberto Simeone HENRIQUES, Adriana Gisele Hertzog da SILVA, Rosário Dominguez Crespo HIRATA, Mario Hiroiuki HIRATA, Sílvia Maria Franciscato COZZOLINOhttps://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessHENRIQUES, Gilberto Simeone SILVA, Adriana Gisele Hertzog da HIRATA, Rosário Dominguez Crespo HIRATA, Mario Hiroiuki COZZOLINO, Sílvia Maria Franciscato 2023-10-05T14:34:16Zoai:ojs.periodicos.puc-campinas.edu.br:article/9990Revistahttp://www.scielo.br/rnPRIhttps://periodicos.puc-campinas.edu.br/nutricao/oai||sbi.submissionrn@puc-campinas.edu.br1678-98651415-5273opendoar:2023-10-05T14:34:16Revista de Nutrição - Pontifícia Universidade Católica de Campinas (PUC-CAMPINAS)false
dc.title.none.fl_str_mv Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats
Transcrição reversa na determinação da expressão do mRNA para a enzima conversora de angiotensina testicular em animais tratados com zinco
title Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats
spellingShingle Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats
HENRIQUES, Gilberto Simeone
diet
angiotensin-converting enzyme
gene expression
reverse transcriptase polymerase chain reaction
zinc
dieta
enzima conversora de angiotensina
expressão gênica
reação em cadeia de polimerase via transcriptase reversa
zinco
title_short Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats
title_full Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats
title_fullStr Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats
title_full_unstemmed Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats
title_sort Assessment of the reverse transcriptase polymerase chain reaction technique in the determination of the mRNA expression for the testicular angiotensin-converting enzyme in zinc treated rats
author HENRIQUES, Gilberto Simeone
author_facet HENRIQUES, Gilberto Simeone
SILVA, Adriana Gisele Hertzog da
HIRATA, Rosário Dominguez Crespo
HIRATA, Mario Hiroiuki
COZZOLINO, Sílvia Maria Franciscato
author_role author
author2 SILVA, Adriana Gisele Hertzog da
HIRATA, Rosário Dominguez Crespo
HIRATA, Mario Hiroiuki
COZZOLINO, Sílvia Maria Franciscato
author2_role author
author
author
author
dc.contributor.author.fl_str_mv HENRIQUES, Gilberto Simeone
SILVA, Adriana Gisele Hertzog da
HIRATA, Rosário Dominguez Crespo
HIRATA, Mario Hiroiuki
COZZOLINO, Sílvia Maria Franciscato
dc.subject.por.fl_str_mv diet
angiotensin-converting enzyme
gene expression
reverse transcriptase polymerase chain reaction
zinc
dieta
enzima conversora de angiotensina
expressão gênica
reação em cadeia de polimerase via transcriptase reversa
zinco
topic diet
angiotensin-converting enzyme
gene expression
reverse transcriptase polymerase chain reaction
zinc
dieta
enzima conversora de angiotensina
expressão gênica
reação em cadeia de polimerase via transcriptase reversa
zinco
description ObjectiveThe aim of the present work was to optimize the reaction conditions capable of generating variability and introducing systematic errors in the chain reaction of the polymerase used to analyze the gene expression for the testicular isoform of the angiotensin-converting enzyme.MethodsThe cDNA concentration, primer concentration, hybridization temperature and number of denaturation, hybridization and extension cycles were evaluated. For this purpose, samples of testis from Wistar rats fed a zinc containing diet were used to extract total RNA using the phenol-chloroform-isothiocyanate reaction. Stable cDNA was then generated by the reverse transcription reaction. Using specific primers, the gene of interest (testicular isoform of the angiotensin-converting enzyme) and the housekeeping gene for the expression of Glyceraldehyde-3-Phosphate Dehydrogenase were amplified. The samples were then submitted to gel eletrophoresis in agarose gel, stained with ethide bromide and visualized in a UV chamber.ResultsThe results showed that the best reaction conditions for the chain reaction by the testicular isoform polymerase of the angiotensin-converting enzyme and for Glyceraldehyde-3-Phosphate Dehydrogenase were: (1) initial cDNA concentration of 2 µg, (2) primer concentration of 200nM, (3) hybridization temperature between 57.5°C and 60.1°C and (4) 33 cycles.ConclusionIt was concluded that this optimization minimized interference of the technique, contributing to the production of true, comparative data for the testicular angiotensin- converting enzyme gene expression.
publishDate 2023
dc.date.none.fl_str_mv 2023-10-05
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://periodicos.puc-campinas.edu.br/nutricao/article/view/9990
url https://periodicos.puc-campinas.edu.br/nutricao/article/view/9990
dc.language.iso.fl_str_mv por
language por
dc.relation.none.fl_str_mv https://periodicos.puc-campinas.edu.br/nutricao/article/view/9990/7327
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 Núcleo de Editoração – PUC-Campinas
publisher.none.fl_str_mv Núcleo de Editoração – PUC-Campinas
dc.source.none.fl_str_mv Brazilian Journal of Nutrition; Vol. 18 No. 6 (2005): Revista de Nutrição
Revista de Nutrição; Vol. 18 Núm. 6 (2005): Revista de Nutrição
Revista de Nutrição; v. 18 n. 6 (2005): Revista de Nutrição
1678-9865
reponame:Revista de Nutrição
instname:Pontifícia Universidade Católica de Campinas (PUC-CAMPINAS)
instacron:PUC_CAMP
instname_str Pontifícia Universidade Católica de Campinas (PUC-CAMPINAS)
instacron_str PUC_CAMP
institution PUC_CAMP
reponame_str Revista de Nutrição
collection Revista de Nutrição
repository.name.fl_str_mv Revista de Nutrição - Pontifícia Universidade Católica de Campinas (PUC-CAMPINAS)
repository.mail.fl_str_mv ||sbi.submissionrn@puc-campinas.edu.br
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