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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: |
null
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Registro original: |
Embrapa Gado de Leite (CNPGL) |
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| Registros recuperados : 6 | |
| 1. |  | MAGALHÃES, L. F.; MAGALHÃES, G. M.; CALAZANS, S. G.; CRIVELLENTI, L. Z.; LIMA, G. P.; MEDEIROS-RONCHI, A. A. Imunomarcação da transição epitélio-mesenquima na neoplasia mamária primária de cadelas e sua metástase em linfonodo. Pesquisa Veterinária Brasileira, Rio de Janeiro, v. 37, n. 12, p. 1479-1482, dezembro 2017. Título em inglês: Epithelial to mesenchymal transition immunostaining in canine primary mammary tumor and lymph node.| Biblioteca(s): Embrapa Unidades Centrais. |
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| 2. | | SOUZA, C. A. de; MAGALHÃES, G. M. de O.; SOUZA, P.; PAZ, E. S.; SANTOS, M. R. A. dos. Padrão de crescimento de calos friáveis de folhas, entrenó e nó de Capsicum annuum var. annuum cv. iberaba Jalapeño. In: JORNADA CIENTÍFICA CEDSA, 10., 2015, Porto Velho. [Anais...]. Porto Velho: Unir, 2015.| Tipo: Artigo em Anais de Congresso |
| Biblioteca(s): Embrapa Rondônia. |
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| 3. | | SANTOS, M. R. A. dos; SOUZA, C. A. de; GUIMARÃES, M. de C. M.; SMOZINSKI, C. V.; MAGALHÃES, G. M. de O.; NOGUEIRA, W. O.; PAZ, E. S. Growth pattern of friable callus from P. Carniconnectivum Leaf explants. Australian Journal of Basic and Applied Sciences, Pakistan, v.9, n. 27, p. 226-231, aug. 2015.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: A - 2 |
| Biblioteca(s): Embrapa Rondônia. |
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| 4. | | GARCIA, M. V.; MAGALHÃES, G. M.; OSHIRO, L. M.; ARDISON, F. de A.; ALMEIDA, R. F. C.; BARROS, J. C.; ANDREOTTI, R. Histopatologia da possível ação do diflubenzuron no controle de Rhipicephalus (B.) microplus. In: INTERNATIONAL SYMPOSIUM ON TICK CONTROL AND TICK-BORNE DISEASES, 2013, Campo Grande, MS. [Proceedings..]. Campo Grande, MS: Embrapa Gado de Corte, 2013. (Embrapa Gado de Corte. Documentos, 202). Comissão organizadora: Renato Andreotti, Fernando Paiva, Wilson Werner Koller, Jacqueline Cavalcante Barros, Luiz Antonio Dias Leal, Jaqueline Matias.| Tipo: Resumo em Anais de Congresso |
| Biblioteca(s): Embrapa Gado de Corte. |
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| 5. | | ROSOLEM, M. C.; VASCONCELOS, R. O.; GARRIDO, E.; CASTANHEIRA, T. L. L.; MOREIRA, P. R. R.; MAGALHÃES, G. M.; ROZZA, D. B.; RAMOS, S. B. Immunodetection of myeloid and plasmacytoid dendritic cells in mammary carcinomas of female dogs. Pesquisa Veterinária Brasileira, Brasília, DF, v. 35, n. 11, p. 906-912, nov. 2015.| Biblioteca(s): Embrapa Unidades Centrais. |
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| 6. | | SANTOS, M. R. A. dos; GUIMARÃES, M. C. M.; PAZ, E. S.; MAGALHÃES, G. M. O.; SOUZA, C. A.; SMOZINSKI, C. V.; NOGUEIRA, W. O. Induction and growth pattern of callus from Piper permucronatum leaves. Revista Brasileira de Plantas Medicinais, Botucatu, v. 18, n. 1, p.142-148, 2016.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: B - 1 |
| Biblioteca(s): Embrapa Rondônia. |
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| Registros recuperados : 6 | |
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