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HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

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RNA Biology, New England Biolabs

 

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Cite this Article: HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

Jani, B., Fuchs, R. In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection. J. Vis. Exp. (61), e3702, doi:10.3791/3702 (2012).

Disclosures: HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

Materials: HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

Name Company Catalog Number Comments
T7 High Yield RNA Synthesis Kit New England Biolabs E2040S
3’-0-Me-m7G(5’)ppp(5’)G RNA Cap Structure Analog New England Biolabs S1411S
DNase I (RNase-free) New England Biolabs M0303S
Vaccinia Capping System New England Biolabs M2080S
MEGAclear Kit Ambion AM1908
NanoDrop Spectrophotometer Thermo Fisher Scientific, Inc.
Agilent RNA 6000 Nano Kit Agilent Technologies 5067-1511
Agilent 2100 Bioanalyzer Agilent Technologies G2939AA
TransIT mRNA Transfection Kit Mirus Bio LLC MIR2250
HyClone* Classical Liquid Media: Dulbecco’s Modified Eagle’s Medium, High Glucose Thermo Fisher Scientific, Inc. SH30285.01
HyClone Non-essential Amino Acids Thermo Fisher Scientific, Inc. SH30238.01
Centro LB 960 Berthold Technologies
BioLux Gaussia Luciferase Assay Kit New England Biolabs E3300S
BioLux Cypridina Luciferase Assay Kit New England Biolabs E3309S
RNase Inhibitor, Murine New England Biolabs M0314
RNase Inhibitor, Human Placenta New England Biolabs M0307
pCMV-GLuc Control Plasmid New England Biolabs N8081

References: HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

  1. Lazarev, D. & Manley J.L. Concurrent splicing and transcription are not sufficient to enhance splicing efficiency. RNA. 13, 1546-1557 (2007).
  2. Donze, O. & Picard, D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA Polymerase. Nucl. Acids Res. 30, 10e46 (2002).
  3. Van Gelder, R.N., et al. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Nati. Acad. Sci. U.S.A. 87, 1663-1667 (1990).
  4. Schneider, J., et al. Systematic analysis of T7 RNA polymerase based in vitro linear RNA amplification for use in microarray experiments. BMC Genomics. 5, 29 (2004).
  5. Conry, R.M., et al. Characterization of a messenger RNA polynucleotide vaccine vector. Cancer Research. 55, 1397-1400 (1995).
  6. Huang, F., Wang, G., Coleman, T., & Li, N. Synthesis of adenosine derivatives as transcription initiators and preparation of 5′ fluorescein- and biotin-labeled RNA through one-step transcription. RNA. 9, 1562-1570 (2003).
  7. Warren, L., et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 7, 618-630 (2010).
  8. Furuichi, Y., LaFiandra, A., & Shatkin, A.J. 5'-Terminal structure and mRNA stability. Nature. 266, 235-239 (1977).
  9. Topisirovic, I., Svitkin, Y.V., Sonenberg, N., & Shatkin, A.J. Cap and cap binding proteins in the control of gene expression. Wiley Interdisciplinary Reviews. 2, 277-298 (2011).
  10. Lewis, J.D. & Izaurflde, E. The Role of the Cap Structure in RNA Processing and Nuclear Export. Eur. J. Biochem. 247, 461-469 (1997).
  11. Inoue, K., Ohno, M., Sakamoto, H., & Shimura, Y. Effect of the cap structure on pre-mRNA splicing in Xenopus oocyte nuclei. Genes Dev. 3, 1472-1479 (1989).
  12. Martin, S.A., Paoletti, E., & Moss, B. Purification of mRNA guanylyltransferase and mRNA (guanine 7-)methyltransferase from vaccinia virus. J. Biol. Chem. 250, 9322-9329 (1975).
  13. Martin, S.A. & Moss, B. Modification of RNA by mRNA guanylytransferase and mRNA (guanine-7-)methyl-transferase from vaccinia virions. J. Biol. Chem. 250, 9330-9335 (1975).
  14. Shuman, S. & Moss, B. Purification and use of vaccinia virus mRNA capping enzyme. Meth. Enzymol. 181, 170-180 (1990).
  15. Thiel, V., Herold, J., Scelle, B., & Siddell, S.G. Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. J. Gen. Virol. 82, 1273-1281 (2001).
  16. Marcotrigiano, J., Gingras, A-C., Sonenberg, N., & Burley, S.K. Cocrystal Structure of the Messenger RNA 5′ Cap-Binding Protein (eIF4E) Bound to 7-methyl-GDP. Cell. 89 (6), 951-961 (1997).
  17. Stepinski, J., et al. Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl (3'-O-methyl)GpppG and 7-methyl (3'-deoxy) GpppG. RNA. 7, 1486-1495 (2001).
  18. Kuge, H., Brownlee, G.G., Gershon, P.D., & Richter, J.D. Cap ribose methylation of c-mos mRNA stimulates translation and oocyte maturation in Xenopus laevis. Nucl. Acids Res. 26 (13), 3208-3214 (1998).
  19. Schagat, T., Paguio, A., & Kopish, K. Normalizing Genetic Reporter Assays: Approaches and Considerations for Increasing Consistency and Statistical Significance. Cell Notes. 17, 9-12 (2007).
  20. Barbosa, E. & Moss, B. mRNA (nucleoside-2'-) - methyltransferase from Vaccinia Virus. J. Biol. Chem. 253 (21), 7698-7702 (1978).
  21. Lockless, S.W., et al. Recognition of capped RNA substrates by VP39, the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase. Biochemistry. 37, 8564-8574 (1998).

Ask the Author: HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

1 Comment

Hi Dr. Jani, how would you quantify the percentage of capped RNA was made from in vitro synthesis set up, which was shown in part 1 protocol of this article ? I assume we do have a mixture of capped and uncapped RNA with in vitro transcription.
Thank you,

1

Reply

Posted by: Thao T.April 4, 2013, 4:52 PM

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