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, 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/10362/163179
Resumo: © 2023 The Authors. Published by Elsevier B.V.
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spelling Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiaeViral dsRNAPhenol extractionAmmonium sulphateRT-PCRSequencingQD ChemistryQR MicrobiologyQH426 GeneticsBiochemistry, Genetics and Molecular Biology (miscellaneous)Molecular BiologySDG 3 - Good Health and Well-beingSDG 9 - Industry, Innovation, and Infrastructure© 2023 The Authors. Published by Elsevier B.V.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.Instituto de Higiene e Medicina Tropical (IHMT)TB, HIV and opportunistic diseases and pathogens (THOP)Global Health and Tropical Medicine (GHTM)Vector borne diseases and pathogens (VBD)RUNCardoso, Fernando M HElias, AlexandreMaurício, IsabelMatos, Olga2024-02-06T23:19:54Z2023-122023-12-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article10application/pdfhttp://hdl.handle.net/10362/163179eng2215-0161PURE: 74939699https://doi.org/10.1016/j.mex.2023.102435info: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:RCAAP2024-05-22T18:18:03Zoai:run.unl.pt:10362/163179Portal AgregadorONGhttps://www.rcaap.pt/oai/openairemluisa.alvim@gmail.comopendoar:71602024-05-22T18:18:03Repositório 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
Viral dsRNA
Phenol extraction
Ammonium sulphate
RT-PCR
Sequencing
QD Chemistry
QR Microbiology
QH426 Genetics
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Molecular Biology
SDG 3 - Good Health and Well-being
SDG 9 - Industry, Innovation, and Infrastructure
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
Maurício, Isabel
Matos, Olga
author_role author
author2 Elias, Alexandre
Maurício, Isabel
Matos, Olga
author2_role author
author
author
dc.contributor.none.fl_str_mv Instituto de Higiene e Medicina Tropical (IHMT)
TB, HIV and opportunistic diseases and pathogens (THOP)
Global Health and Tropical Medicine (GHTM)
Vector borne diseases and pathogens (VBD)
RUN
dc.contributor.author.fl_str_mv Cardoso, Fernando M H
Elias, Alexandre
Maurício, Isabel
Matos, Olga
dc.subject.por.fl_str_mv Viral dsRNA
Phenol extraction
Ammonium sulphate
RT-PCR
Sequencing
QD Chemistry
QR Microbiology
QH426 Genetics
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Molecular Biology
SDG 3 - Good Health and Well-being
SDG 9 - Industry, Innovation, and Infrastructure
topic Viral dsRNA
Phenol extraction
Ammonium sulphate
RT-PCR
Sequencing
QD Chemistry
QR Microbiology
QH426 Genetics
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Molecular Biology
SDG 3 - Good Health and Well-being
SDG 9 - Industry, Innovation, and Infrastructure
description © 2023 The Authors. Published by Elsevier B.V.
publishDate 2023
dc.date.none.fl_str_mv 2023-12
2023-12-01T00:00:00Z
2024-02-06T23:19:54Z
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/10362/163179
url http://hdl.handle.net/10362/163179
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 2215-0161
PURE: 74939699
https://doi.org/10.1016/j.mex.2023.102435
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 10
application/pdf
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 mluisa.alvim@gmail.com
_version_ 1817545985056833536

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