Simplicon™ RNA Frequently Asked Questions

What is the maximum size of inserts that can be cloned into the SimpliconCloning Vector (E3L)? (Cat. #: SCR724)

The maximum size of inserts that can be accommodated is ~8,300 bp at least. For example, Human OKSG-cMyc TagRFP Simplicon™ plasmid (Cat# SCR729) contains 8,301 bp of insert in Simplicon™ plasmid. The expected Simplicon™ RNA size of Human OKSG-cMyc TagRFP Simplicon™ is ~17.5 kb.

How much and how long does Simplicon™ RNA express?

In general, small sized insert can express much more proteins. Expression of the transgenes will decrease depending on size and numbers of insert gene(s).  Duration of expression depends on cell types, insert genes, and culture conditions. In our experience, Human OKSG-cMyc TagRFP Simplicon™ RNA can continuously express the TagRFP in human foreskin fibroblasts with DMEM/10% FBS medium (HFF culture medium) containing B18R proteins and puromycin, while most iPSCs colonies generated with Human OKSG-cMyc TagRFP Simplicon™ RNA (ES culture medium containing B18R and puromycin) lose the TagRFP expression in 15 days. If you are using a recombinant B18R protein, a recombinant B18R protein from HEK293 (GF197) will work for long term sustained expression.

Is phosphatase treatment required for Simplicon™ RNA?

Phosphatase treatment is required for Simplicon™ RNA with three-step RNA synthesis (Enzymatic capping method). If Simplicon™ RNA is generated with one-step RNA synthesis using CleanCap AG reagent, it is not necessary.

How long can a Simplicon™ RNA remain stable?

  1. Simplicon™ RNA is stable for at least 1 year in RNA storage solution at -80 °C.  Avoid freeze thaws.
  2. The RNA pellet after 2.5 M ammonium acetate precipitation is stable for several months at -80°C. Therefore, it is possible to stop the reaction of RNA synthesis after 2.5 M ammonium acetate. Always wash the RNA pellet with 70% ethanol to remove residual ammonium acetate, and then store the RNA pellet at -80°C until restart.

Are modified nucleotides required for RNA synthesis?

It is not necessary to use modified nucleotides such as 5-Methyl-CTP and Pseudo-UTP for either the Simplicon™ and B18R-E3L RNA syntheses. When modified nucleotides are used, do not replace more than 25% of CTP and UTP. 100% replacement of CTP and UTP with modified nucleotides will lose the expression of Simplicon™ RNA. Higher replacement will decrease the protein expression of B18R-E3L RNA.  

Which Simplicon™ product is right for you?

Product Name Cat. No. Application
Simplicon™ Plasmids
Simplicon Cloning Vector (E3L) SCR724 To synthesize Simplicon™ RNA in vitro containing the transgene(s) of interest. At least a total of 8.3kb of transgene(s) can be synthesized into Simplicon™ RNA, followed by successful high sustained protein expression in transfected human cell lines, without the risk of genome integration.
TagGFP2 Simplicon™ Plasmid (E3L) SCR725 To determine optimal transfection conditions to express the self-replicating RNA of your interest through using Simplicon Cloning Vector (E3L) (Part #:SCR724) in hard-to- transfect somatic or primary cells.
TagRFP Simplicon™ Plasmid (E3L) SCR726 The TagRFP Simplicon™ Plasmid (E3L) was developed for the synthesis of TagRFP Simplicon™ RNA (E3L). The Simplicon™ TagRFP RNA may be used to determine optimal transfection conditions to express the self-replicating RNA in hard-to transfect somatic or primary cells.
Human OKSG-cMyc TagRFP Simplicon™ Plasmid SCR729 To produce unlimited amount of Human OKSG-cMyc TagRFP Simplicon™ RNA for human iPSCs generation along with a red fluorescent protein (TagRFP).
Simplicon™ RNAs
Fluorescence Tag Simplicon™ RNAs
TagGFP2 Simplicon™ RNA (E3L) Kit SCR720 To determine optimal transfection conditions to express the self-replicating RNA of your interest through using Simplicon™ Cloning Vector (E3L) (Part #:SCR724) in hard-to- transfect somatic or primary
TagRFP Simplicon™ RNA (E3L) Kit SCR721 To monitor and optimize the transfection of experiment Simplicon™ RNAs into the human cell line of interest.
Human Simplicon™ Reprogramming RNAs
Simplicon™ Reprogramming RNA (OKSG) SCR549 To generate integration free, virus-free human iPS cell using a single transfection step of self-replicating synthetic RNA (Simplicon™ RNA) containing Oct4, Klf4, Sox2, and Glis1.
Simplicon™ RNA Reprogramming Kit (OKSG) SCR550 To generate integration free, virus-free human iPS cell using a single transfection step of self-replicating synthetic RNA (Simplicon™ RNA) containing Oct4, Klf4, Sox2, and Glis1. Kit includes Human Recombinant B18R Protein and Human iPS Reprogramming Boost Supplement II.
Human OKSG-cMyc Simplicon RNA SCR703 To generate integration free, virus-free human iPS cell using a single transfection step of self-replicating synthetic RNA (Simplicon™ RNA) containing Oct4, Klf4, Sox2, Glis1 and cMyc. The 5-factor OKSG-cMyc Simplicon is especially useful for iPSCs generation from somatic cells that are more difficult to reprogram (i.e. slower proliferating cells or aged somatic cells).
Human OKSG-cMyc TagRFP Simplicon RNA SCR714 The OKSG-cMyc transgenes are especially useful for iPSCs generation from somatic cells that are more difficult to reprogram (i.e. slower proliferating cells or aged somatic cells) while the TagRFP provides a rapid assessment of transfection efficiency. Presence of the TagRFP transgene also allows for optimization of the transfection conditions in hard- to- transfect somatic or primary cells.
B18R Plasmids, RNAs and Proteins
B18R Plasmids
B18R-E3L Plasmid (human codon optimized for B18R and E3L) SCR727 To strongly suppress more interferon responses than single B18R to enable more expression of a Simplicon™ RNA or regular mRNA(s).
B18R Plasmid (human codon optimized) SCR728 To supply B18R RNA that suppresses IFN responses to support Simplicon™ RNA or normal mRNA expression.
B18R RNAs
B18R-E3L RNA (human codon optimized for B18R and E3L) SCR722 To suppress the strong interferon (IFN) response elicited by the introduction and replication of the Simplicon™ RNA in the transfected human cells.
B18R RNA (human codon optimized) SCR723 Required for high expression of Simplicon™ RNA at transfection and available for production of B18R-CM (conditioned medium).
B18R Proteins
Vaccinia Virus B18R protein, recombinant expressed in HEK 293 cells, Carrier-Free GF197 To suppress the strong interferon (IFN) response elicited by the introduction and replication of the Simplicon™ RNA in the transfected human cells and enable more sustained transgene(s) expression.

General References:

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Liu, Z., et al. (2017). Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7(1): 2193.

Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in mammalian cells. J Virol 79(12): 7597-7608.Petrakova O,

Pegg G., et al. (2018). Design and development of metabolically competent cell lines for high throughput, chemical compound, toxicity testing, Drug Metabolism and Pharmacokinetics 33 (1): S33-S34

Perdiguero B, Esteban M. (2009). The interferon system and vaccinia virus evasion mechanisms. J Interferon Cytokine Res. 29(9): 581-98.

Volkova E, Gorchakov R, Paessler S, Kinney RM, Frolov I (2005).

Yoshioka N, Dowdy SF (2017). Enhanced generation of iPSCs from older adult human cells by a synthetic five-factor self-replicative RNA. PLoS One. 12(7): e0182018.

Yoshioka N, Gros E, Li HR, Kumar S, Deacon DC, Maron C, Muotri AR, Chi NC, Fu XD, Yu BD, Dowdy SF (2013). Efficient generation of human iPSCs by a synthetic self-replicative RNA. Cell Stem Cell. 13 (2): 246-254.

Zimmer G (2010). RNA replicons – a new approach for influenza virus immunoprophylaxis. Viruses 2(2): 413-434.