Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Journal of Health & Biological Sciences |
Texto Completo: | https://periodicos.unichristus.edu.br/jhbs/article/view/2562 |
Resumo: | Background: RNA isolation from bacteria within dentine caries lesions could be difficult due to reduced amount of collectable biomass and high mRNA instability. Attempting to overcome this challenge we describe one protocol developed to extract and purify total RNA from dentine lesions. Objective: customize a bacterial RNA extraction and purification method from human carious dentine. Methods: quantity and purity of extracted RNA were measured with a microvolume UV-VIS spectrophotometer, RNA integrity was assessed by standard denaturing agarose gel electrophoresis and images were captured under ultraviolet light with camera and analyzed. DNase treatment removed genomic DNA and an additional step of purification was carried out in silica spin column. Results: final yield (ng/μl) was 67.01 ± 22.33, absorbance ratio A260/A280 = 2.0 ± 0.07 and RNA integrity were obtained. The purified samples were reversely transcribed and the expression of atpD and fabM gene from Streptococcus mutans analyzed by quantitative real-time PCR. Conclusion: the extraction methodology developed produced high-quality RNA from dentine microbiota for transcriptional analysis. |
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Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studiesCiências Biológicas;Odontologia;Ciências da SaúdeDentine Caries; RNA Extraction; Gene Expression; Streptococcus mutansMolecular biologyBackground: RNA isolation from bacteria within dentine caries lesions could be difficult due to reduced amount of collectable biomass and high mRNA instability. Attempting to overcome this challenge we describe one protocol developed to extract and purify total RNA from dentine lesions. Objective: customize a bacterial RNA extraction and purification method from human carious dentine. Methods: quantity and purity of extracted RNA were measured with a microvolume UV-VIS spectrophotometer, RNA integrity was assessed by standard denaturing agarose gel electrophoresis and images were captured under ultraviolet light with camera and analyzed. DNase treatment removed genomic DNA and an additional step of purification was carried out in silica spin column. Results: final yield (ng/μl) was 67.01 ± 22.33, absorbance ratio A260/A280 = 2.0 ± 0.07 and RNA integrity were obtained. The purified samples were reversely transcribed and the expression of atpD and fabM gene from Streptococcus mutans analyzed by quantitative real-time PCR. Conclusion: the extraction methodology developed produced high-quality RNA from dentine microbiota for transcriptional analysis.Instituto para o Desenvolvimento da EducacaoThis study was supported by CNPqBezerra, Daniela da SilvaNeves, Beatriz GonçalvesGuedes, Sarah Florindo de FigueiredoRegis, Wanessa Fernandes MatiasStipp, Rafael NobregaRodrigues, Lidiany Karla Azevedo2019-04-11info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionAvaliado por ParesPeer ReviewRevisado por paresapplication/pdfhttps://periodicos.unichristus.edu.br/jhbs/article/view/256210.12662/2317-3076jhbs.v7i2.2562.p145-151.2019Journal of Health & Biological Sciences; v. 7, n. 2(Abr-Jun) (2019): Journal of Health and Biological Sciences; 145-151Journal of Health & Biological Sciences; v. 7, n. 2(Abr-Jun) (2019): Journal of Health and Biological Sciences; 145-151Journal of Health and Biological Sciences; v. 7, n. 2(Abr-Jun) (2019): Journal of Health and Biological Sciences; 145-1512317-30762317-308410.12662/2317-3076jhbs.v7i2.2019reponame:Journal of Health & Biological Sciencesinstname:Centro Universitário Christus (Unichristus)instacron:CHRISTUSenginfo:eu-repo/grantAgreement/EC/FP7/475346https://periodicos.unichristus.edu.br/jhbs/article/view/2562/834Mundialinfo:eu-repo/semantics/openAccessDireitos autorais 2019 Journal of Health & Biological Scienceshttp://creativecommons.org/licenses/by-nc-sa/4.02019-07-01T20:15:44Zoai:ojs.unichristus.emnuvens.com.br:article/2562Revistahttps://periodicos.unichristus.edu.br/jhbs/indexPRIhttps://periodicos.unichristus.edu.br/jhbs/oaisecretaria.jhbs@unichristus.edu.br || editor.jhbs@fchristus.edu.br2317-30762317-3084opendoar:2023-01-13T09:47:20.629636Journal of Health & Biological Sciences - Centro Universitário Christus (Unichristus)true |
dc.title.none.fl_str_mv |
Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies |
title |
Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies |
spellingShingle |
Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies Bezerra, Daniela da Silva Ciências Biológicas;Odontologia;Ciências da Saúde Dentine Caries; RNA Extraction; Gene Expression; Streptococcus mutans Molecular biology |
title_short |
Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies |
title_full |
Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies |
title_fullStr |
Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies |
title_full_unstemmed |
Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies |
title_sort |
Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies |
author |
Bezerra, Daniela da Silva |
author_facet |
Bezerra, Daniela da Silva Neves, Beatriz Gonçalves Guedes, Sarah Florindo de Figueiredo Regis, Wanessa Fernandes Matias Stipp, Rafael Nobrega Rodrigues, Lidiany Karla Azevedo |
author_role |
author |
author2 |
Neves, Beatriz Gonçalves Guedes, Sarah Florindo de Figueiredo Regis, Wanessa Fernandes Matias Stipp, Rafael Nobrega Rodrigues, Lidiany Karla Azevedo |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
This study was supported by CNPq |
dc.contributor.author.fl_str_mv |
Bezerra, Daniela da Silva Neves, Beatriz Gonçalves Guedes, Sarah Florindo de Figueiredo Regis, Wanessa Fernandes Matias Stipp, Rafael Nobrega Rodrigues, Lidiany Karla Azevedo |
dc.subject.por.fl_str_mv |
Ciências Biológicas;Odontologia;Ciências da Saúde Dentine Caries; RNA Extraction; Gene Expression; Streptococcus mutans Molecular biology |
topic |
Ciências Biológicas;Odontologia;Ciências da Saúde Dentine Caries; RNA Extraction; Gene Expression; Streptococcus mutans Molecular biology |
description |
Background: RNA isolation from bacteria within dentine caries lesions could be difficult due to reduced amount of collectable biomass and high mRNA instability. Attempting to overcome this challenge we describe one protocol developed to extract and purify total RNA from dentine lesions. Objective: customize a bacterial RNA extraction and purification method from human carious dentine. Methods: quantity and purity of extracted RNA were measured with a microvolume UV-VIS spectrophotometer, RNA integrity was assessed by standard denaturing agarose gel electrophoresis and images were captured under ultraviolet light with camera and analyzed. DNase treatment removed genomic DNA and an additional step of purification was carried out in silica spin column. Results: final yield (ng/μl) was 67.01 ± 22.33, absorbance ratio A260/A280 = 2.0 ± 0.07 and RNA integrity were obtained. The purified samples were reversely transcribed and the expression of atpD and fabM gene from Streptococcus mutans analyzed by quantitative real-time PCR. Conclusion: the extraction methodology developed produced high-quality RNA from dentine microbiota for transcriptional analysis. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-04-11 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Avaliado por Pares Peer Review Revisado por pares |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://periodicos.unichristus.edu.br/jhbs/article/view/2562 10.12662/2317-3076jhbs.v7i2.2562.p145-151.2019 |
url |
https://periodicos.unichristus.edu.br/jhbs/article/view/2562 |
identifier_str_mv |
10.12662/2317-3076jhbs.v7i2.2562.p145-151.2019 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/grantAgreement/EC/FP7/475346 https://periodicos.unichristus.edu.br/jhbs/article/view/2562/834 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess Direitos autorais 2019 Journal of Health & Biological Sciences http://creativecommons.org/licenses/by-nc-sa/4.0 |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
Direitos autorais 2019 Journal of Health & Biological Sciences http://creativecommons.org/licenses/by-nc-sa/4.0 |
dc.format.none.fl_str_mv |
application/pdf |
dc.coverage.none.fl_str_mv |
Mundial |
dc.publisher.none.fl_str_mv |
Instituto para o Desenvolvimento da Educacao |
publisher.none.fl_str_mv |
Instituto para o Desenvolvimento da Educacao |
dc.source.none.fl_str_mv |
Journal of Health & Biological Sciences; v. 7, n. 2(Abr-Jun) (2019): Journal of Health and Biological Sciences; 145-151 Journal of Health & Biological Sciences; v. 7, n. 2(Abr-Jun) (2019): Journal of Health and Biological Sciences; 145-151 Journal of Health and Biological Sciences; v. 7, n. 2(Abr-Jun) (2019): Journal of Health and Biological Sciences; 145-151 2317-3076 2317-3084 10.12662/2317-3076jhbs.v7i2.2019 reponame:Journal of Health & Biological Sciences instname:Centro Universitário Christus (Unichristus) instacron:CHRISTUS |
instname_str |
Centro Universitário Christus (Unichristus) |
instacron_str |
CHRISTUS |
institution |
CHRISTUS |
reponame_str |
Journal of Health & Biological Sciences |
collection |
Journal of Health & Biological Sciences |
repository.name.fl_str_mv |
Journal of Health & Biological Sciences - Centro Universitário Christus (Unichristus) |
repository.mail.fl_str_mv |
secretaria.jhbs@unichristus.edu.br || editor.jhbs@fchristus.edu.br |
_version_ |
1797052856279236608 |