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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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| Registros recuperados : 16 | |
| 6. |  | ARAÚJO, L. da S.; COSTA, E. M. R.; SOARES, T. L.; SANTOS, I. S. dos; JESUS, O. N. de. Effect of time and storage conditions on the physical and physico-chemical characteristics of the pulp of yellow and purple passion fruit. Food Science and Technology, Campinas, v.37, n.3, p. 500-506, July-Sept. 2017.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: B - 1 |
| Biblioteca(s): Embrapa Mandioca e Fruticultura. |
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| 8. | | ARAÚJO, L. DA S.; JESUS, O. N. de; COSTA, E. M. R.; SOARES, T. L.; SANTOS, I. S. DOS; ARAÚJO, D. M. S. Avaliação química de frutos de maracujá de casca amarela e roxa em diferentes condições e período de armazenamento. In: CONGRESSO BRASILEIRO DE RECURSOS GENÉTICOS, 3., 2014, Santos. Anais... Brasília, DF: Sociedade Brasileira de Recursos Genéticos, 2014.| Tipo: Resumo em Anais de Congresso |
| Biblioteca(s): Embrapa Mandioca e Fruticultura. |
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| 9. | | ARAÚJO, L. DA S.; JESUS, O. N. de; COSTA, E. M. R.; SOARES, T. L.; SANTOS, I. S, DOS; ARAÚJO, D. M. S. Caracterização de acessos e híbridos de maracujazeiro por meio de descritores morfológicos. In: CONGRESSO BRASILEIRO DE RECURSOS GENÉTICOS, 3., 2014, Santos. Anais... Brasília, DF: Sociedade Brasileira de Recursos Genéticos, 2014.| Tipo: Resumo em Anais de Congresso |
| Biblioteca(s): Embrapa Mandioca e Fruticultura. |
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| 11. | | NETTO, M. de S.; OLIVEIRA, F. C. de; ARAÚJO, L. da S.; SILVEIRA, P. M. da; CUNHA, P. C. R. da. Épocas, formas de aplicação e doses de nitrogênio na cultura do milho em condições de cerrado. Colloquium Agrariae, v. 16, n. 6, p. 56-66, nov./dez. 2020.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: B - 3 |
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| 12. | | ARAÚJO, L. da S.; SILVA, L. G. B.; VALENTE, M. S.; CORREIA, N. M.; NASCIMENTO, W. M.; AQUINO, L. C. de; CUNHA, P. C. R. da. Seletividade de herbicidas aplicados em pós-emergência para a cultura do grão de bico. In: CONGRESSO BRASILEIRO DA CIÊNCIA DAS PLANTAS DANINHAS, 30., 2016, Curitiba. Conhecimento e tecnologia a serviço do agricultor: anais. Curitiba: Sociedade Brasileira da Ciência das Plantas Daninhas, 2016. p. 514.| Biblioteca(s): Embrapa Hortaliças. |
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| 13. | | ARAÚJO, L. da S.; SILVA, G. S.; SILVA, L. G. B.; VALENTE, M. S.; AQUINO, L. C. de; CORREIA, N. M.; CUNHA, P. C. R. da. Seletividade de sulfentrazone e trifluralina aplicados em pré-emergência para a cultura do feijão adzuki. In: CONGRESSO BRASILEIRO DA CIÊNCIA DAS PLANTAS DANINHAS, 30., 2016, Curitiba. Conhecimento e tecnologia a serviço do agricultor: anais. Curitiba: Sociedade Brasileira da Ciência das Plantas Daninhas, 2016. p. 401.| Biblioteca(s): Embrapa Hortaliças. |
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| 14. | | SILVA, L. G. B.; ARAÚJO, L. da S.; GONÇALVES, D. J.; VALENTE, M. S.; SILVA, A. R. da; NASCIMENTO, W. M.; CUNHA, P. C. R. da. Selectivity of diphenyl-ether herbicides with postemergence applications in chickpea. Journal of Plant Protection Research, v. 59, n. 3, p. 350-354, 2019.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: B - 1 |
| Biblioteca(s): Embrapa Hortaliças. |
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| 15. | | ARAÚJO, L. da S.; CUNHA, P. C. R. da; SILVEIRA, P. M. da; SOUSA NETTO, M. de; OLIVEIRA, F. C. de. Potencial de cobertura do solo e supressão de tiririca (Cyperus rotundus ) por resíduos culturais de plantas de cobertura. Revista Ceres, Viçosa, MG, v. 62, n. 5, p. 483-488, set./out. 2015.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: B - 1 |
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| 16. | | SILVA, F. H. da; CUNHA, P. C. R. da; ALMEIDA, A. C. de S.; ARAÚJO, L. da S.; JAKELAITIS, A.; SILVEIRA, P. M. da. Production components of corn as function of seed distribution along the planting row. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande, v. 19, n. 12, p. 1172-1177, dez. 2015.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: A - 2 |
| Biblioteca(s): Embrapa Arroz e Feijão. |
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| Registros recuperados : 16 | |
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| Nenhum registro encontrado para a expressão de busca informada. |
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