Simplicon™ RNA Transfection and Electroporation Protocols

Tranfsection of Simplicon™ RNAs has been validated using the RiboJuice^™ mRNA Transfection Kit and Lipofectamine^® MessengerMAX™ Transfection Reagent. Amounts of RNAs and transfection reagents may vary depending on the target cells. Set up different RNA: transfection reagent ratios.

A) Forward Transfection Protocol

1. Plate target cells to reach 50-90% confluency at time of transfection in 6-well pate. Set aside an untransfected control well to observe the puromycin cell death. Sensitivity to puromycin may vary with different cell types.
2. Wash cells once with DMEM (no serum, no antibiotics) and add 1 mL/well of DMEM (no serum, no antibiotics).
   

a. Option 1: Add 200 ng/mL of B18R protein (MilliporeSigma GF156 or Sigma GF197) in medium. Pre-treatment of B18R protein may support the neutralization of IFN responses. Incubate cells at 37 °C in a CO2 incubator for 10-20 minutes with B18R protein.

b. Option 2: No serum condition increases the transfection efficiency. However, it is possible to use 1-10% serum depending upon cell types.

1. Set up transfection reactions in sterile eppendorf tubes. Follow order of additions. Mix gently by pipetting during each addition of RNA and reagent. Do not vortex.

If using MessengerMAX™ Transfection Reagent (ThermoFisher LMRNA001):

Total RNA Amount		1 µg	2 µg
1) Prepare RNA mixture in Tube 1.
Tube 1	DMEM (no serum, no antibiotics)	50 µL	100 µL
	B18R-E3L RNA (1 µg/ µL)	0.5 µL	1 µL
	Simplicon™ RNA (1 µg/ µL)	0.5 µL	1 µL
	Total volume	51 µL	102 µL
2) Prepare MessengerMAX™ dilution mixture in Tube 2. No incubation! Incubation of MessengerMAX™ dilution significantly decreases the transfection efficiency.
Tube 2	DMEM (no serum, no antibiotics)	50 µL	100 µL
	MessengerMAX™	3 µL	6 µL
	Total volume	53 µL	106 µL
3) Quickly add tube 2 into tube 1.
Total volume in a tube		104 µL	208 µL

If using RiboJuice™ mRNA Transfection Kit (Part No. TR-1013):

Component	Vial	Cat. No.
Opti-MEM®	250 µL	ThermoFisher (31985-062)
B18R-E3L RNA (1 µg/µL)	0.5 µL
Simplicon™ RNA (1µg/µL)	0.5 µL
RiboJuice™ mRNA Boost Reagent	4.0 µL	RiboJuice™ mRNA Transfection (TR-1013)
RiboJuice™ mRNA Transfection Reagent	4.0 µL
Total Volume	259 µL

4. Incubate at room temperature for 5 minutes and add the RNA-transfection reagent complex dropwise into one well of the 6-well plate containing cells.
5. Incubate the plate at 37 ˚C in a CO₂ incubator for 2-4 hrs. Maximum transfection efficiency is obtained with 4 hours incubation using MessengerMAX™ reagent in human fibroblasts.
6. Aspirate the transfection medium and add 2 mL per well of culture medium containing 10-20% of B18R-CM or 200 ng/mL B18R protein. Incubate at 37 °C in a CO₂ incubator overnight.
7. Next day, aspirate and exchange with fresh culture medium containing 10-20% of B18R-CM or 200 ng/mL B18R protein and puromycin (0.2-1 µg/mL). B18R protein and puromycin should be added fresh each time. Puromycin selection is used to remove cells that have not taken up the Simplicon™ RNA. Sensitivity to puromycin may vary with different cell types and must be determined empirically.
8. Change medium every day for sustaining expression of Simplicon^TM RNA. Add fresh 10-20% of B18R-CM or 200 ng/mL B18R protein and puromycin with each media change. In general, puromycin selection works in 5 days. For long term expression of Simplicon™ RNA after a week, it is possible to transition to media changes every other day and also to reduce the amounts of puromycin (0.1-0.5 µg/mL) and 10-20% of B18R-CM or B18R protein (50-200 ng/mL).
9. Analyze cells depending on experiments.
   
   Note: Some IFNs may not be neutralized by B18R protein and will accumulate in the medium. Cell passaging will remove IFNs more efficiently as compared to media changes and will also help with the long-term expression of the Simplicon™ RNA.

B) Reverse Transfection Protocol

For some cells (i.e. HepG2) reverse transfection may be more efficient.

1. Prepare target cells to reach 80-100% confluent at the time of transfection.
2. On the day of transfection, detach cells with cell detachment solution such as AccuMax, Accutase or Trypsin/EDTA to make a single cell suspension. Collect cells in regular cell culture medium.
3. Briefly centrifuge to pellet the cells. Aspirate medium. Resuspend cells in normal culture medium containing 2% serum and 200 ng/mL B18R protein (no antibiotics) and transfer cells to a new well to achieve 50-100% the next day.
   
   Note: The percentage of serum in the resuspension medium is dependent on cell types. In general, low serum condition will increase the transfection efficiency.
   
   
4. Place newly plated cells at 37 °C in a CO₂ incubator while you prepare the RNA transfection mixture as previously outlined in step 3 of the Forward Transfection Protocol.
5. Follow Steps 4–9 of the Forward Transfection Protocol.

C) Electroporation for primary T cells (human)

Electroporation is an alternative way to introduce Simplicon^™ RNA into difficult to transfect cells such as primary human T cells (activated and expanded from peripheral blood mononuclear cells (PBMCs)). Using this protocol, it is possible to achieve 20-70% electroporation efficiency of Simplicon™ TagGFP2 RNA to primary human T cells.

1. Prepare healthy growing primary human T cells. We generally use CD3/CD28 beads (ThermoFisher 11131D) and rIL2 (MilliporeSigma IL002) for activation and expansion of primary human T cells.
2. Using a Bio-Rad electroporation unit, set up the following program: exponential pulse condition of 160V and 950 µF.
3. Prepare 1x10⁶ cells in Ingenio^® Electroporation Solution (Mirus Bio MIR50111) in 100 uL total volume. Transfer cell mixture to an electroporation cuvette (0.2 cm-gap). Store on ice.
4. Prepare RNA mixture: 5 µg of Simplicon™ RNA plus 5 µg of B18R-E3L RNA.
5. Add RNAs mixture to cells. RNAs are not stable after mixing with cells.
6. Do electroporation as soon as possible.
7. Put on ice for a few minutes, and then transfer the cells into appropriate wells of a 48 well plate with 1 mL of cell culture medium containing 200 ng/mL B18R protein.
8. Incubate at 37 °C in a CO₂ incubator overnight.
9. Assay protein expression of Simplicon™ RNA using flow analysis or ICC.
10. For continuous expression of the Simplicon™ RNA, B18R protein or B18R-CM and puromycin should be present throughout the duration of the cell culture experiment. B18R protein and B18R-CM and puromycin should be added fresh during media changes. Puromycin selection is used to remove cells that have not taken up the Simplicon™ RNA. Sensitivity to puromycin may vary with different cell types and must be determined empirically.

Representative Data of Transfection Experiments:

In the Simplicon™ RNA Expression System, a new IFN suppressor, E3L, was introduced as a polycistronic B18R-E3L RNA to suppress the IFN responses at RNA transfection and also incorporated into Simplicon™ RNA itself to suppress the IFN responses during RNA replication. B18R-E3L RNA worked better than B18R RNA at the transfection with Simplicon™ TagGFP2 (Figure 1A). Addition of E3L into Simplicon™ RNA increased the expression of Simplicon™ TagRFP in sustained expression (Figure 1B). Simplicon™ RNA can be introduced with RNA transfection or RNA electroporation in many types of cells as indicated (Figure 1C).

B18R-E3L RNA is also available for mRNA transfection to suppress the IFN responses. B18R-E3L RNA worked better than B18R-RNA in mRNA transfection (Figure 2A). Suppression of IFN responses at mRNA transfection enables the repeated transfection of mRNA (Figure 2B).

Figure 1. (A) E3L increased SimpliconTM RNA expression levels. BJ human foreskin fibroblasts were co-transfected with SimpliconTM TagGFP2 and B18R RNA or B18R-E3L RNA. (B) E3L worked for continuous expression of SimpliconTM RNA. SimpliconTM TagRFP or TagRFP Simplicon (E3L) was co-transfected with B18R-E3L RNA and cultured with medium containing B18R protein and puromycin for 14 days. RFP expressing cells were imaged on Day 12, and analyzed by FACS on Day 14. (C) SimpliconTM RNA can be transfected into a wide variety of cell types. Simplicon™ TagGFP2 RNA and B18R-RNA were co-transfected with Human iPSCs, LX2 human hepatic stellate cell line (MilliporeSigma SCC064), human mesenchymal stem cells (MSCs, MilliporeSigma SCC034) by MessengerMAX™ transfection reagent. For human primary T cells (PBMCs stimulated with CD3/CD28), electroporation method was used.

Figure 2. B18R-E3L or B18R RNA for mRNA transfection. (A) BJ human foreskin fibroblasts were co-transfected with TagGFP2 mRNA and B18R RNA or B18R-E3L RNA. (B) B18R or B18R-E3L RNA co-transfection enables repeated transfection of mRNA. BJ cells were transfected with TagGFP2 mRNA Plus/Minus B18R RNA or B18R-E3L RNA at 1st transfection (upper panel, 1st transfection). Next day, TagRFP mRNA was transfected into the same cells, and imaged for RFP expression one day after the transfection (bottom panel, 2nd transfection). The mRNAs for TagGFP2 and TagRFP were synthesized without modified nucleotides.