Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UFLA |
Texto Completo: | http://repositorio.ufla.br/jspui/handle/1/39370 |
Resumo: | The Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative. |
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Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorumAjuste de protocolos melhora a extração de RNA total de alta qualidade de hastes de feijão infectadas por Sclerotinia sclerotiorumPhaseolus vulgaris L.RNA isolationCommon bean - White moldPlant-pathogen interactionIsolamento de RNAFeijão comum - Mofo brancoThe Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative.O Straw Test é um ensaio desenvolvido para avaliar a resistência do feijoeiro ao mofo branco, no qual as hastes da planta são inoculados e os sintomas da doença são monitorados. É plausível admitir que a investigação da expressão gênica em tecidos infectados por patógenos possa ser estrategicamente interessante. No entanto, obter um RNA de qualidade é um requisito básico para essa finalidade. Portanto, o objetivo deste estudo foi avaliar ajustes em protocolos de kits comerciais na expectativa de melhorar a qualidade do RNA obtido a partir de hastes de feijão. Para tanto, plantas de duas linhagenslinees foram inoculadas e as hastes infectadas pelo patógeno foram coletadas após 72 horas. Para extração de RNA, dois reagentes comerciais foram utilizados inicialmente seguindo as recomendações do fabricante e seguindo as adaptações desses protocolos. Em especial, as modificações propostas referem-se aos volumes de sobrenadante recuperados nos passos de purificação, etapa adicional de purificação por clorofórmio e maior tempo para precipitação dos ácidos nucleicos. O RNA obtido foi analisado por espectrofotômetro, eletroforese e bioanalyzer, posteriormente convertido em cDNA e, por fim, submetido à PCR. A partir dos dados obtidos, observou-se que as adaptações feitas nos protocolos contribuíram para resultados melhores e que quando os valores indicativos de qualidade do RNA são garantidos, as reações subsequentes são mais puras, precisas e representativas.Universidade Federal de Lavras2020-03-25T13:47:26Z2020-03-25T13:47:26Z2019info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfMIRANDA, R. N. de; SILVA, C. M. da; PORTO, A. C. da M.; PEREIRA, W. A. Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum. Ciência e Agrotecnologia, Lavras, v. 43, p. 1-10, 2019. DOI: https://doi.org/10.1590/1413-7054201943024618 .http://repositorio.ufla.br/jspui/handle/1/39370Ciência e Agrotecnologiareponame:Repositório Institucional da UFLAinstname:Universidade Federal de Lavras (UFLA)instacron:UFLAAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessMiranda, Rafael Novais deSilva, Caroline Marcela daPorto, Antonio Carlos da MotaPereira, Welison Andradeeng2020-03-25T13:47:26Zoai:localhost:1/39370Repositório InstitucionalPUBhttp://repositorio.ufla.br/oai/requestnivaldo@ufla.br || repositorio.biblioteca@ufla.bropendoar:2020-03-25T13:47:26Repositório Institucional da UFLA - Universidade Federal de Lavras (UFLA)false |
dc.title.none.fl_str_mv |
Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum Ajuste de protocolos melhora a extração de RNA total de alta qualidade de hastes de feijão infectadas por Sclerotinia sclerotiorum |
title |
Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum |
spellingShingle |
Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum Miranda, Rafael Novais de Phaseolus vulgaris L. RNA isolation Common bean - White mold Plant-pathogen interaction Isolamento de RNA Feijão comum - Mofo branco |
title_short |
Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum |
title_full |
Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum |
title_fullStr |
Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum |
title_full_unstemmed |
Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum |
title_sort |
Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum |
author |
Miranda, Rafael Novais de |
author_facet |
Miranda, Rafael Novais de Silva, Caroline Marcela da Porto, Antonio Carlos da Mota Pereira, Welison Andrade |
author_role |
author |
author2 |
Silva, Caroline Marcela da Porto, Antonio Carlos da Mota Pereira, Welison Andrade |
author2_role |
author author author |
dc.contributor.author.fl_str_mv |
Miranda, Rafael Novais de Silva, Caroline Marcela da Porto, Antonio Carlos da Mota Pereira, Welison Andrade |
dc.subject.por.fl_str_mv |
Phaseolus vulgaris L. RNA isolation Common bean - White mold Plant-pathogen interaction Isolamento de RNA Feijão comum - Mofo branco |
topic |
Phaseolus vulgaris L. RNA isolation Common bean - White mold Plant-pathogen interaction Isolamento de RNA Feijão comum - Mofo branco |
description |
The Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019 2020-03-25T13:47:26Z 2020-03-25T13:47:26Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
MIRANDA, R. N. de; SILVA, C. M. da; PORTO, A. C. da M.; PEREIRA, W. A. Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum. Ciência e Agrotecnologia, Lavras, v. 43, p. 1-10, 2019. DOI: https://doi.org/10.1590/1413-7054201943024618 . http://repositorio.ufla.br/jspui/handle/1/39370 |
identifier_str_mv |
MIRANDA, R. N. de; SILVA, C. M. da; PORTO, A. C. da M.; PEREIRA, W. A. Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum. Ciência e Agrotecnologia, Lavras, v. 43, p. 1-10, 2019. DOI: https://doi.org/10.1590/1413-7054201943024618 . |
url |
http://repositorio.ufla.br/jspui/handle/1/39370 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.rights.driver.fl_str_mv |
Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Attribution 4.0 International http://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 |
Universidade Federal de Lavras |
publisher.none.fl_str_mv |
Universidade Federal de Lavras |
dc.source.none.fl_str_mv |
Ciência e Agrotecnologia reponame:Repositório Institucional da UFLA instname:Universidade Federal de Lavras (UFLA) instacron:UFLA |
instname_str |
Universidade Federal de Lavras (UFLA) |
instacron_str |
UFLA |
institution |
UFLA |
reponame_str |
Repositório Institucional da UFLA |
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Repositório Institucional da UFLA |
repository.name.fl_str_mv |
Repositório Institucional da UFLA - Universidade Federal de Lavras (UFLA) |
repository.mail.fl_str_mv |
nivaldo@ufla.br || repositorio.biblioteca@ufla.br |
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1807835174653657088 |