Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/27614 |
Resumo: | RNA quality is of utmost importance to perform gene expression quantification by qPCR. The classical methods used to determine \RNA\ quality are based on electrophoresis and spectrophotometer assessment, namely A260/A280 and A260/A230 ratios. It was previously shown that due to the complex nature of Staphylococcus epidermidis biofilms, \RNA\ extraction procedures could impact mRNA quality and thus accurate quantification. Herein, we contaminated and degraded \RNA\ extracted from S. epidermidis biofilms, and assessed the effect on gene expression by qPCR. As expected, thermal degradation of RNA had a significant impact on gene expression on two out of the three tested genes. On the other hand, the contamination of the extracted \RNA\ yielded an interesting result: while most contaminants did not changed the purity indicators or the integrity of RNA, significant changes on gene expression levels were found. This work confirms that poor \RNA\ extraction has an important impact in qPCR quantification, emphasizing the consequences of carry-over contaminants on gene expression studies. Additionally, our results show that the parameters commonly used to assess the quality of extracted \RNA\ from bacterial cultures seem to be insufficient to ensure reliable gene expression determination. |
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Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilmsS. epidermidis biofilmRNA qualityqPCRImpuritiesemperature degradationTemperature degradationScience & TechnologyRNA quality is of utmost importance to perform gene expression quantification by qPCR. The classical methods used to determine \RNA\ quality are based on electrophoresis and spectrophotometer assessment, namely A260/A280 and A260/A230 ratios. It was previously shown that due to the complex nature of Staphylococcus epidermidis biofilms, \RNA\ extraction procedures could impact mRNA quality and thus accurate quantification. Herein, we contaminated and degraded \RNA\ extracted from S. epidermidis biofilms, and assessed the effect on gene expression by qPCR. As expected, thermal degradation of RNA had a significant impact on gene expression on two out of the three tested genes. On the other hand, the contamination of the extracted \RNA\ yielded an interesting result: while most contaminants did not changed the purity indicators or the integrity of RNA, significant changes on gene expression levels were found. This work confirms that poor \RNA\ extraction has an important impact in qPCR quantification, emphasizing the consequences of carry-over contaminants on gene expression studies. Additionally, our results show that the parameters commonly used to assess the quality of extracted \RNA\ from bacterial cultures seem to be insufficient to ensure reliable gene expression determination.This work was funded by the Fundacao para a Ciencia e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309. The following authors had an individual FCT fellowship: VC (SFRH/BD/78235/2011); LDRM (SFRH/BD/66166/2009).ElsevierElsevier BVUniversidade do MinhoCarvalhais, Virgínia Maria DinisDelgado-Rastrollo, M.Melo, Luís D. R.Cerca, Nuno20132013-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/27614eng0167-70120167-701210.1016/j.mimet.2013.08.01023999274info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T11:58:12Zoai:repositorium.sdum.uminho.pt:1822/27614Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T18:47:54.598200Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms |
title |
Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms |
spellingShingle |
Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms Carvalhais, Virgínia Maria Dinis S. epidermidis biofilm RNA quality qPCR Impurities emperature degradation Temperature degradation Science & Technology |
title_short |
Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms |
title_full |
Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms |
title_fullStr |
Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms |
title_full_unstemmed |
Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms |
title_sort |
Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms |
author |
Carvalhais, Virgínia Maria Dinis |
author_facet |
Carvalhais, Virgínia Maria Dinis Delgado-Rastrollo, M. Melo, Luís D. R. Cerca, Nuno |
author_role |
author |
author2 |
Delgado-Rastrollo, M. Melo, Luís D. R. Cerca, Nuno |
author2_role |
author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Carvalhais, Virgínia Maria Dinis Delgado-Rastrollo, M. Melo, Luís D. R. Cerca, Nuno |
dc.subject.por.fl_str_mv |
S. epidermidis biofilm RNA quality qPCR Impurities emperature degradation Temperature degradation Science & Technology |
topic |
S. epidermidis biofilm RNA quality qPCR Impurities emperature degradation Temperature degradation Science & Technology |
description |
RNA quality is of utmost importance to perform gene expression quantification by qPCR. The classical methods used to determine \RNA\ quality are based on electrophoresis and spectrophotometer assessment, namely A260/A280 and A260/A230 ratios. It was previously shown that due to the complex nature of Staphylococcus epidermidis biofilms, \RNA\ extraction procedures could impact mRNA quality and thus accurate quantification. Herein, we contaminated and degraded \RNA\ extracted from S. epidermidis biofilms, and assessed the effect on gene expression by qPCR. As expected, thermal degradation of RNA had a significant impact on gene expression on two out of the three tested genes. On the other hand, the contamination of the extracted \RNA\ yielded an interesting result: while most contaminants did not changed the purity indicators or the integrity of RNA, significant changes on gene expression levels were found. This work confirms that poor \RNA\ extraction has an important impact in qPCR quantification, emphasizing the consequences of carry-over contaminants on gene expression studies. Additionally, our results show that the parameters commonly used to assess the quality of extracted \RNA\ from bacterial cultures seem to be insufficient to ensure reliable gene expression determination. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013 2013-01-01T00:00:00Z |
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 |
http://hdl.handle.net/1822/27614 |
url |
http://hdl.handle.net/1822/27614 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
0167-7012 0167-7012 10.1016/j.mimet.2013.08.010 23999274 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Elsevier Elsevier BV |
publisher.none.fl_str_mv |
Elsevier Elsevier BV |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
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1799132238690910208 |