Simplicon™ RNA Troubleshooting Tips

No or Low RNA yield

  1. Check DNA template: Check the amount, quality and size of the linearized DNA (single digestion). Contamination of phenol & chloroform will reduce the activity of the T7 RNA polymerase. Wash DNA pellet with 70% ethanol to remove salts and impurities.
  2. Check transcription reaction mixture: Make sure that all of the components are included in the RNA transcription mixture. 5x buffer may have a precipitate after thawing; ensure that 5x buffer is fully resuspended and that the correct amount is added to the transcription reaction mixture. 
  3. T7 RNA polymerase:  The T7 promoter sequence in the Simplicon™ plasmid has been optimized for expression of Simplicon™ RNA. Not all T7 RNA Polymerase will work effectively in the Simplicon™ RNA synthesis.  We recommend the use of T7 RNA polymerase from Promega (Cat. No. P1300).

RNA degradation is observed

  1. Use RNase-free reagents and tips.
  2. Use high quality (transfection grade) DNA plasmid and freshly prepared DNA template for the RNA transcription reaction.
  3. Because Simplicon™ RNA is a large sized RNA, some amount of degradation is expected as shown in the RNA electrophoresis gel on pg. 22. 
  4. Reaction time of RNA transcription:  2 hours reaction time is optimal for minimizing RNA degradation while still yielding high amounts of RNA.  One hour incubation may be tried, but RNA yields may be lower.
  5. Use RNA storage solution (ThermoFisher AM7000) for long-term storage of RNA at -80°C. TE buffer or water should not be used.

Significant RNA loss after 2.5 M ammonium acetate precipitation

  1. A 10-20% loss of RNA may be expected with 2.5 M ammonium acetate precipitation.
  2. The quality of 5 M ammonium acetate may affect the RNA yields. Store 5 M ammonium acetate at 2-8°C to maintain high quality.
  3. Alternatively, RNA purification columns are available for RNA purification.

Transfection Problems

  1. MessengerMax (ThermoFisher LNRNA001) can work for a broad range of cell types. While adult keratinocytes can be transfected using MessengerMax, neonatal keratinocytes are more difficult to transfect.  Different type of transfection reagents and method may be used such as RiboJuice™ mRNA Transfection Kit (MilliporeSigma TR-1013,), or electroporation with Ingenio® Electroporation Solution (Mirus Bio MIR 50111). In general, lipid based DNA transfection reagent such as Lipofectamine2000 can work for Simplicon™ RNA transfection.
  2. Increase the amount of transfection reagent per µg RNA.  Determine empirically the optimal amount of transfection reagent to apply while still minimizing cytotoxicity.
  3. In general, the size of transgenes along with the number of transgenes to be expressed will have an impact on expression levels and transfection efficiency.  The smaller size of the transgenes will get the higher the expression levels and transfection efficiency.
  4. Dead cells are observed after transfection.

a.  Reduce the amount of RNAs to be transfected

b.  Try a different transfection reagent.

c.  Pretreat with B18R protein before and after transfection.

No protein expression

  1. B18R-E3L RNA is required for expression of Simplicon™ RNA at transfection. Do quality check of B18R-E3L RNA by co-transfection with Simplicon™ TagGFP2 (MilliporeSigma SCR720) or TagRFP (MilliporeSigma SCR721).
  2. Check DNA sequence of Simplicon™ plasmid: Confirm DNA sequence of insert gene(s) and 26S subgenomic promoter region that is required for transcription of insert gene(s).
  3. In general, the size of transgenes along with the number of transgenes to be expressed will have an impact on expression levels and transfection efficiency.  The smaller size of the transgenes will get the higher the expression levels and transfection efficiency.  

No continuous expression

  1. Do puromycin selection: It is better to remove un-transfected cells by puromycin selection. High doses of puromycin selection may kill all the cells. Simplicon™ RNA expression is high for a few days after transfection, and becomes stable at low levels in a week.  Puromycin is located after the second IRES and the last gene of the Simplicon™ RNA. Therefore, puromycin selection can be performed at very low amount of puromycin (0.2-1 µg/mL; mostly 0.2-0.4 µg/mL) for 5-7 days selection.
  2. B18R is needed for continuous expression. Change cell culture medium every day and freshly supply B18R conditioned medium (CM) or B18R protein. If you are using a recombinant B18R protein, a recombinant B18R protein from HEK293 (Sigma GF197) will work for long term sustained expression.
  3. Continuous expression is dependent on cell types, insert genes, and culture conditions.

No good quality of B18R conditioned medium

  1. Highly proliferative fibroblasts such as Human Foreskin Fibroblasts (MilliporeSigma SCC058) is recommended for production of B18R-CM. At least, HFFs (MilliporeSigma SCC058) is more proliferative and expressed more proteins compared to BJ fibroblasts.
  2. B18R-CM can work for expression of Simplicon™ RNA, but weaker compared to the co-  transfection with B18R-E3L RNA.
  3. Check the quality of B18R-E3L RNA as described in “Quality Check II:  RNA Transfection”.
  4. Passage HFFs one day before the B18R-E3L RNA transfection. Condition of HFFs is important to get high transfection efficiency and protein production. Transfection efficiency of HFFs will decrease in a few days after passaging.

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.