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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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| Registros recuperados : 13 | |
| 2. |  | ALFENAS, R. F.; PEREIRA, O. L.; FREITAS, R. G.; FREITAS, C. S.; RODRIGUEZ, M. A. D.; ALFENAS, A. C. Mass spore production and inoculation of Calonectria pteridis on Eucalyptus spp. under different environmental conditions. Tropical Plant Pathology, v. 38, n.5, p. 406-413, 2013.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: B - 1 |
| Biblioteca(s): Embrapa Mandioca e Fruticultura. |
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| 4. | | FREITAS, R. G.; MISSIO, R. F.; MATOS, F. S.; RESENDE, M. D. V. de; DIAS, L. A. S. Genetic evaluation of Jatropha curcas: an important oilseed for biodiesel production. Genetics and Molecular Research, Ribeirão Preto, v. 10, n. 3, p. 1490-1498, 2011.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: B - 1 |
| Biblioteca(s): Embrapa Florestas. |
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| 5. | | FREITAS, R. G. de; VASCONCELOS, E. S. de; CRUZ, C. D.; ROSADO, A. M.; ROCHA, R. B.; TAKAMI, L. K. Predição de ganhos genéticos em progênies de polinização aberta de Eucalyptus urograndis cultivadas em diferentes ambientes e submetidas a diferentes procedimentos de seleção. Revista Árvore, Viçosa, MG, v. 33, n. 2, mar./abr. 2009. doi: 10.1590/S0100-67622009000200007| Tipo: Artigo em Periódico Indexado | Circulação/Nível: B - 1 |
| Biblioteca(s): Embrapa Rondônia. |
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| 6. | | CARDOSO, P. M. R.; DIAS, L. A. dos S.; RESENDE, M. D. V. de; FREITAS, R. G. de; CORRÊA, T. R.; MUNIZ, D. R.; ZAIDAN, I. R. Genetic evaluation and selection in Jatropha curcas L. Crop Breeding and Applied Biotechnology, Viçosa, MG, v. 18, n. 2, p. 192-199, Apr./June 2018.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: A - 2 |
| Biblioteca(s): Embrapa Florestas. |
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| 7. | | FREITAS, R. G. de; MATOS, F. S.; SILVA, D. F. da; MISSIO, R. F.; RESENDE, M. D. V. de; DIAS, L. A. dos S. Diversidade genética em acessos de Jatropha curcas L. através de variáveis canônicas. In: CONGRESSO DA REDE BRASILEIRA DE TECNOLOGIA DE BIODIESEL, 4.; CONGRESSO BRASILEIRO DE PLANTAS OLEAGINOSAS, ÓLEOS, GORDURAS E BIODIESEL, 7., 2010, Belo Horizonte. Biodiesel: inovação tecnológica e qualidade: anais: trabalhos científicos. Lavras: UFLA, 2010.| Tipo: Artigo em Anais de Congresso |
| Biblioteca(s): Embrapa Florestas. |
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| 8. | | GONDIM, T. C. O.; DIAS, L. A. S.; DIAS, D. C. F. S.; LAVIOLA, B. G.; ANDRADE, V. M.; OLIVEIRA, G. L.; FREITAS, R. G.; RIBEIRO, R. M.; MARTINS, A. O. Avaliação do desenvolvimento de mudas de pinhão manso produzidas por estacas em diferentes substratos. In: CONGRESSO DA REDE BRASILEIRA DE TECNOLOGIA DE BIODIESEL, 3., 2009, Brasília, DF. Anais... Brasília, DF: MCT, 2009.| Tipo: Artigo em Anais de Congresso / Nota Técnica |
| Biblioteca(s): Embrapa Agroenergia. |
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| 9. | | GONDIM, T. C. de O.; DIAS, L. A. dos S.; DIAS, D. C. F. dos S.; LAVIOLA, B. G.; FREITAS, R. G. de; SILVA, L. J. da; MARTINS, A. O. Características agronômicas e produção inicial do pinhão manso proveniente de plantas oriundas de estacas de diferentes tamanhos ou de sementes. In: CONGRESSO DA REDE BRASILEIRA DE TECNOLOGIA DE BIODIESEL, 4.; CONGRESSO BRASILEIRO DE PLANTAS OLEAGINOSAS, ÓLEOS, GORDURAS E BIODIESEL, 7., 2010, Belo Horizonte. Biodiesel: inovação tecnológica e qualidade: anais. Lavras: UFLA, 2010. v. 2. p. 733-734. il.| Tipo: Artigo em Anais de Congresso |
| Biblioteca(s): Embrapa Agroenergia. |
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| 10. | | PEREIRA, F. R. da S.; REIS, A. A. dos; FREITAS, R. G.; OLIVEIRA, S. R. de M.; AMARAL, L. R. do; FIGUEIREDO, G. K. D. A.; ANTUNES, J. F. G.; LAMPARELLI, R. A. C.; MORO, E.; MAGALHÃES, P. S. G. Imputation of missing parts in UAV orthomosaics using PlanetScope and Sentinel-2 data: a case study in a grass-dominated área. International Journal of Geo-Information, v. 12, n. 2, 41, Feb. 2023.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: A - 2 |
| Biblioteca(s): Embrapa Agricultura Digital. |
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| 11. | | SPADA, E. R.; PEREIRA, E. A.; MONTANHERA, M. A.; MORAIS, L. H.; FREITAS, R. G.; COSTA, R. G. F.; SOARES, G. B.; OLIVEIRA, C. R. de; PAULA, F. R. Preparation, characterization and application of phase-pure anatase and rutile TiO2, nanoparticles by new green route. In: Journal of Materials Science: Materials in Electronics, v. 28, p. 16932-16938, 2017.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: A - 1 |
| Biblioteca(s): Embrapa Instrumentação. |
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| 12. | | GONDIM, T. C. de O.; DIAS, L. A. dos S.; DIAS, D. C. F. dos S.; LAVIOLA, B. G.; OLIVEIRA, G. L.; FREITAS, R. G. de; RIBEIRO, R. da M.; MARTINS, A. O. Desenvolvimento de mudas de pinhão manso propagadas por sementes e por estacas de tamanho reduzido. In: CONGRESSO BRASILEIRO DE PLANTAS OLEAGINOSAS, ÓLEOS, GORDURAS E BIODIESEL, 6., 2009, Montes Claros, MG. Biodiesel: inovação tecnológica – anais. Lavras: UFLA, 2009.| Tipo: Artigo em Anais de Congresso / Nota Técnica |
| Biblioteca(s): Embrapa Agroenergia. |
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| 13. | | HERMENEGILDO, P. da S.; FREITAS, R. G. de; CASCARDO, R. de S; GUIMARÃES, L. M. S.; BADEL, J. L.; ALFENAS‑ZERBINI, P.; MARQUES, A. S. dos A.; ALFENAS, A. C.; FERREIRA, M. A. S. V. Sensitive detection, quantification, and monitoring of Erwinia psidii colonization of guava plants using intercalating dye-based real-time PCR. Tropical Plant Pathology, v. 48, p. 375-383, 2022. Na publicação: Abi S. A. Marques.| Tipo: Artigo em Periódico Indexado | Circulação/Nível: A - 2 |
| Biblioteca(s): Embrapa Recursos Genéticos e Biotecnologia. |
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| Registros recuperados : 13 | |
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| Nenhum registro encontrado para a expressão de busca informada. |
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