Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae

Detalhes bibliográficos
Autor(a) principal: Cardoso, Fernando M.H.
Data de Publicação: 2023
Outros Autores: Elias, Alexandre, Pereira, Inês, Maurício, Isabel, Matos, Olga
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10451/59990
Resumo: © 2023 Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
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spelling Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiaeAmmonium sulphateImproved dsRNA isolation and purification- phase partitionPhenol extractionRT-PCRSequencingViral dsRNA© 2023 Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)Accurate genomic sequencing demands high-quality double-stranded RNA (dsRNA). Existing methods for dsRNA extraction from yeast, fungi, and plants primarily rely on cellulose, suitable only for small volume extractions, or the time-consuming lithium chloride precipitation. To streamline the traditional phenol-chloroform-based dsRNA extraction method, the main challenge is the reduction of mitochondrial DNA (mtDNA) and Single Stranded RNA (ssRNA) to no detectable levels after gel electrophoresis. This challenge is successfully addressed through the modified approach described here, involving phenol extraction at low pH, followed by the addition of ammonium sulfate to the aqueous buffer. The dsRNA isolated using this novel method exhibits comparable quality to that obtained through cellulose purification, and it is readily amenable to RT-PCR. Moreover, a single batch of yeast cell RNA isolation requires only 2-3 h of hands-on time, thus simplifying and expediting the process significantly.•Buffers were redesigned from [32,33,35].•No DNASE, Ribonuclease A or beads were used during the purification.•Simple and inexpensive dsRNA extraction and purification method is described.This study was partially supported by FCT/MCTES for supporting Research Units GHTM - UID/04413/2020 and LA-REAL-LA/P/0117/2020.ElsevierRepositório da Universidade de LisboaCardoso, Fernando M.H.Elias, AlexandrePereira, InêsMaurício, IsabelMatos, Olga2023-10-26T10:54:57Z20232023-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10451/59990engMethodsX. 2023 Oct 11:11:10243510.1016/j.mex.2023.1024352215-0161info: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:RCAAP2023-11-08T17:09:32Zoai:repositorio.ul.pt:10451/59990Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T22:09:49.626852Repositório 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 Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae
title Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae
spellingShingle Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae
Cardoso, Fernando M.H.
Ammonium sulphate
Improved dsRNA isolation and purification- phase partition
Phenol extraction
RT-PCR
Sequencing
Viral dsRNA
title_short Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae
title_full Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae
title_fullStr Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae
title_full_unstemmed Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae
title_sort Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae
author Cardoso, Fernando M.H.
author_facet Cardoso, Fernando M.H.
Elias, Alexandre
Pereira, Inês
Maurício, Isabel
Matos, Olga
author_role author
author2 Elias, Alexandre
Pereira, Inês
Maurício, Isabel
Matos, Olga
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Cardoso, Fernando M.H.
Elias, Alexandre
Pereira, Inês
Maurício, Isabel
Matos, Olga
dc.subject.por.fl_str_mv Ammonium sulphate
Improved dsRNA isolation and purification- phase partition
Phenol extraction
RT-PCR
Sequencing
Viral dsRNA
topic Ammonium sulphate
Improved dsRNA isolation and purification- phase partition
Phenol extraction
RT-PCR
Sequencing
Viral dsRNA
description © 2023 Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
publishDate 2023
dc.date.none.fl_str_mv 2023-10-26T10:54:57Z
2023
2023-01-01T00:00:00Z
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/10451/59990
url http://hdl.handle.net/10451/59990
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv MethodsX. 2023 Oct 11:11:102435
10.1016/j.mex.2023.102435
2215-0161
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
publisher.none.fl_str_mv Elsevier
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
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str 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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