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 | Acesso ao texto completo restrito à biblioteca da Embrapa Gado de Leite. Para informações adicionais entre em contato com cnpgl.biblioteca@embrapa.br. |
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Registro Completo |
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Biblioteca(s): |
Embrapa Gado de Leite. |
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Data corrente: |
26/12/2018 |
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Data da última atualização: |
24/01/2023 |
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Tipo da produção científica: |
Artigo em Periódico Indexado |
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Autoria: |
SOUZA, G. T. de; HELL, R. C. R.; SOUZA, J. F. da S.; CAMARGO, L. S. de A. |
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Afiliação: |
LUIZ SERGIO DE ALMEIDA CAMARGO, CNPGL. |
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Título: |
Easy in vitro synthesis of optimised functioning reporter mRNA from common eGFP plasmid. |
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Ano de publicação: |
2018 |
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Fonte/Imprenta: |
Molecular Biotechnology, v. 60, n. 10, p. 762-771, 2018. |
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DOI: |
10.1007/s12033-018-0112-5 |
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Idioma: |
Inglês |
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Conteúdo: |
Abstract The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which has been observed as difficult-to-transfect DNA constructs. eGFP expression could be detected both by flow cytometry and by fluorescence microscopy after transfection with the polyadenylated mRNAs. Upon cytometric analysis, we noted a marked difference among the mRNA groups (p < 0.01), both in fluorescent population percentage and in florescence intensity. We showed here the necessity of the polyadenylation step in order to achieve cell expression of the eGFP observable under fluorescence microscopy. The presence of the Kozak sequence, as a 5' element, seems to augment significantly the level of protein produced upon mRNA transfection. We presented here an easy protocol to allow production of functioning mRNAs from any DNA construct. The molecules produced may aid in the standardisation and controlling most of the RNA-related experiments as well as it gives proper guidance for researchers performing expression of other proteins through mRNA transfection. MenosAbstract The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which ... Mostrar Tudo |
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Palavras-Chave: |
CRISPR/Cas9; EGFP; GFP; Kozak; MRNA; Sequence. |
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Thesagro: |
RNA. |
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Categoria do assunto: |
X Pesquisa, Tecnologia e Engenharia |
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Marc: |
LEADER 03181naa a2200253 a 4500
001 2102521
005 2023-01-24
008 2018 bl uuuu u00u1 u #d
024 7 $a10.1007/s12033-018-0112-5$2DOI
100 1 $aSOUZA, G. T. de
245 $aEasy in vitro synthesis of optimised functioning reporter mRNA from common eGFP plasmid.$h[electronic resource]
260 $c2018
520 $aAbstract The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which has been observed as difficult-to-transfect DNA constructs. eGFP expression could be detected both by flow cytometry and by fluorescence microscopy after transfection with the polyadenylated mRNAs. Upon cytometric analysis, we noted a marked difference among the mRNA groups (p < 0.01), both in fluorescent population percentage and in florescence intensity. We showed here the necessity of the polyadenylation step in order to achieve cell expression of the eGFP observable under fluorescence microscopy. The presence of the Kozak sequence, as a 5' element, seems to augment significantly the level of protein produced upon mRNA transfection. We presented here an easy protocol to allow production of functioning mRNAs from any DNA construct. The molecules produced may aid in the standardisation and controlling most of the RNA-related experiments as well as it gives proper guidance for researchers performing expression of other proteins through mRNA transfection.
650 $aRNA
653 $aCRISPR/Cas9
653 $aEGFP
653 $aGFP
653 $aKozak
653 $aMRNA
653 $aSequence
700 1 $aHELL, R. C. R.
700 1 $aSOUZA, J. F. da S.
700 1 $aCAMARGO, L. S. de A.
773 $tMolecular Biotechnology$gv. 60, n. 10, p. 762-771, 2018.
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Registro original: |
Embrapa Gado de Leite (CNPGL) |
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Biblioteca |
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Origem |
Tipo/Formato |
Classificação |
Cutter |
Registro |
Volume |
Status |
URL |
Voltar
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Registro Completo
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Biblioteca(s): |
Embrapa Arroz e Feijão. |
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Data corrente: |
14/01/2009 |
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Data da última atualização: |
18/01/2013 |
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Tipo da produção científica: |
Artigo em Anais de Congresso / Nota Técnica |
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Autoria: |
SOARES, D. M.; AIDAR, H.; THUNG, M.; KLUTHCOUSKI, J. |
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Afiliação: |
DINO MAGALHAES SOARES, CNPAF; HOMERO AIDAR, CNPAF; MICHAEL THUNG, Consultor CNPAF; JOAO KLUTHCOUSKI, CNPAF. |
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Título: |
Importância e necessidade de alimentos em geral, no Brasil, com ênfase no feijão: produção e consumo |
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Ano de publicação: |
2008 |
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Fonte/Imprenta: |
In: CONGRESSO NACIONAL DE PESQUISA DE FEIJÃO, 9., 2008, Campinas. Ciência e tecnologia na cadeia produtiva do feijão. Campinas: Instituto Agronômico, 2008. |
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Descrição Física: |
1 CD-ROM. |
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Série: |
(IAC. Documentos, 85). |
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Idioma: |
Português |
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Conteúdo: |
O objetivo deste trabalho é analisar sucintamente a produção de alimentos em geral, principalmente, do feijão no Brasil, em 2007 e 2008, conforme IBGE/LSPA de julho de 2008, e o respectivo consumo com base nos dados do IBGE e do DIEESE, para entender melhor o consumo per capita anual de feijão. |
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Palavras-Chave: |
CONAFE. |
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Thesagro: |
Consumo; Feijão; Phaseolus Vulgaris; Produção. |
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Categoria do assunto: |
-- |
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URL: |
https://ainfo.cnptia.embrapa.br/digital/bitstream/item/74786/1/193.pdf
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Marc: |
LEADER 01048nam a2200229 a 4500
001 1205039
005 2013-01-18
008 2008 bl uuuu u00u1 u #d
100 1 $aSOARES, D. M.
245 $aImportância e necessidade de alimentos em geral, no Brasil, com ênfase no feijão$bprodução e consumo
260 $aIn: CONGRESSO NACIONAL DE PESQUISA DE FEIJÃO, 9., 2008, Campinas. Ciência e tecnologia na cadeia produtiva do feijão. Campinas: Instituto Agronômico$c2008
300 $c1 CD-ROM.
490 $a(IAC. Documentos, 85).
520 $aO objetivo deste trabalho é analisar sucintamente a produção de alimentos em geral, principalmente, do feijão no Brasil, em 2007 e 2008, conforme IBGE/LSPA de julho de 2008, e o respectivo consumo com base nos dados do IBGE e do DIEESE, para entender melhor o consumo per capita anual de feijão.
650 $aConsumo
650 $aFeijão
650 $aPhaseolus Vulgaris
650 $aProdução
653 $aCONAFE
700 1 $aAIDAR, H.
700 1 $aTHUNG, M.
700 1 $aKLUTHCOUSKI, J.
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Registro original: |
Embrapa Arroz e Feijão (CNPAF) |
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