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Choosing the Right Transfection Reagent for Optimal Efficiency

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Abstract

Optimal conditions must be determined experimentally when transfecting cultured cells. An ideal reagent should offer high transfection efficiency and low toxicity across a broad range of cell types. Here, we compare several reagents for transfecting mammalian cells and offer guidelines for optimizing transfection conditions.

Brad Hook and Amy Landreman Promega Corporation Publication Date: November 2018

Introduction

Several variables are involved when transfecting nucleic acids into mammalian cells. Cell health, growth stage and confluency can all have significant impact on overall transfection efficiency. Selecting the right transfection reagent is also an important consideration, and the reagent:DNA ratio and total amount of DNA transfected typically require optimization in order to achieve the best results. The popularity of lipid-based transfection reagents is largely due to their ease of use and high efficiency when compared to other transfection methods, such as electroporation. However, even with these reagents, transfection conditions must be optimized with each new cell line or type of nucleic acid. Not all reagents will work well with all cell types.

Here, we report the results of a transfection trial with three Promega reagents and another widely used transfection reagent. Each reagent was tested with a range of transfection reagent:DNA ratios and two concentrations of DNA. We also provide some tips for optimizing transfection conditions in cultured cells.

 

Materials and Methods

Transfection Conditions

Cells were grown in the recommended medium for each cell line. For adherent cell lines, 1 x 104 cells were plated in 96-well plates and grown overnight. For suspension cell lines, 2 x 104 cells were plated in 96-well plates on the day of transfection.

Transfection reagents were prepared at room temperature following the directions for each reagent. The DNA used was pGL4.13[luc2/SV40], a plasmid that expresses a modified firefly luciferase gene optimized for mammalian cell expression, under the control of the SV40 promoter.

The following reagent to DNA ratios were tested according to the manufacturer’s instructions:

ViaFect™ Reagent: 4:1, 3:1, 2.5:1 and 2:1

FuGENE® 6 Reagent: 6:1, 4:1, 3:1 and 1.5:1

FuGENE® HD Reagent: 4:1, 3:1, 2.5:1 and 2:1

Lipofectamine® 2000: 5:1, 4:1, 3:1 and 2:1

Each reagent-DNA mixture was pipetted onto 6 wells of cells in either 5µl or 10µl volumes. One 96-well plate/transfection reagent was used per cell line. Plates were mixed on a plate shaker for 10–15 seconds. They were transferred to a 37°C/5% CO2 incubator for 24 hours prior to assaying for cell viability and luciferase reporter activity. Wells with cells only were used as a 100% viability control.

Cell Viability and Transfection Efficiency

Cell viability was determined 24 hours after transfection using the CellTiter-Fluor™ Cell Viability Assay (Cat.# G6080), a non-lytic, single-reagent-addition fluorescence assay based on measurement of a conserved and constitutive protease activity within live cells. After reading fluorescence, ONE-Glo™ Luciferase Assay Reagent was added to the cells, followed by incubation for 10 minutes at room temperature. Luminescence was measured to determine firefly luciferase activity, and the percent transfection efficiency was calculated.

 

Results and Discussion

Transfection Reagent Comparisons

Table 1 provides a summary of the transfection study. Transfection was assessed through expression of firefly luciferase (EXP). The best condition (highest relative luminescence units [RLUs]) was chosen for each transfection reagent. For each cell line, the maximum RLUs were set at 100% for one reagent and the other three values were judged relative to that reading. Cells were also assayed for cell viability (VIA) and compared to a no-transfection control for each cell line. Transfection efficiencies lower than 50% were not reported. ViaFect™ Reagent provided optimal transfection efficiency and low toxicity for the majority of the cell lines tested, although there were some cell lines (e.g., K562 and RAW 264.7) where Lipofectamine® 2000 reagent performed better.

Table 1. Summary of the transfection reagent comparison.

Cell Line FuGENE® 6 FuGENE® HD ViaFect™ L2K
  EXP VIA EXP VIA EXP VIA EXP VIA
A549 ++ ++ +++ +++ +++ +++    
C2C12         +++ +++    
CHO ++ +++     +++ ++ ++ ++
COS7 +++ +++ +++ +++ +++ +++ +++ +++
H9C2         +++ +++ ++ +++
HCT116     +++ ++ +++ +++ ++ ++
HEK 293 ++ ++     +++ +++    
HeLa     +++ +++ ++ +++    
HepG2         +++ ++    
HT-29         +++ +++ +++ +++
Huh7         +++ ++ ++ ++
Jurkat         +++ +++ ++ +++
K562             +++ +++
LNCaP         +++ +++    
MCF7         +++ +++ +++ +++
NIH 3T3         ++ +++ +++ +++
PC-12     ++ +++ +++ +++ +++ +++
PC-3     +++ +++ +++ +++    
RAW 264.7 +++ ++ +++ ++     ++ ++
U2OS     ++ +++ +++ +++    
+++ >80% of max RLUs; >80% of cells were viable ++ >50% to <80% max RLUs; >50% to <80% of cells were viable
EXP, transfection efficiency based on expression of the transfected luciferase gene; VIA, cell viability assessed by the CellTiter-Fluor™ Cell Viability Assay compared to a no-transfection control; L2K, Lipofectamine® 2000.

transfection reagent comparison viafect fugene lipofectamine HeLa cells 15220ma-w

Figure 1. Comparison of transfection reagents in HeLa cells. Cells were transfected at the indicated reagent:DNA ratios, and transfection efficiency and cell viability were determined 24 hours after transfection as described in the methods section.

Figure 1 shows an example of the assay results obtained for HeLa cells. In this experiment, all three Promega reagents exhibited low toxicity (as judged by cell viability percentages) across the range of reagent:DNA ratios tested, with FuGENE® HD providing the highest efficiency. While Lipofectamine® 2000 reagent demonstrated transfection efficiencies only slightly lower those of ViaFect™ Reagent, it was considerably more toxic to HeLa cells at all tested ratios of reagent to DNA.

Table 2 examines transfection efficiency and cell viability under optimal conditions for each reagent 24 hours after transfection. Transfection efficiency was determined through expression of firefly luciferase. The conditions that produced the highest RLUs for each transfection reagent were compared relative to the reagent that produced the highest overall RLUs (MAX). The percent of RLU Max is reported along with the optimal reagent:DNA ratio and whether 50ng (5µl) or 100ng (10µl) produced the highest RLUs for that reagent. Viability was judged relative to untransfected control cells. Details for luciferase expression below 50% of MAX RLUs were not reported.

Table 2. Reagent comparison under optimal conditions for each cell line.

Cell Line Measure FuGENE® 6 FuGENE® HD ViaFect™ L2K
A549 % RLU Max (ratio; vol.) 60% (4:1; 10µl) 90% (3:1; 5µl) MAX (4:1; 10µl) 30%
% Viable @ 24hr >90% >90% >90% 60%
C2C12 % RLU Max (ratio; vol.) <10% <10% MAX (4:1; 10µl) 25%
% Viable @ 24hr >90% >90% >90% >90%
CHO % RLU Max (ratio; vol.) 60% (6:1; 5µl) 30% MAX (4:1; 10µl) 65% (2:1; 10µl)
% Viable @ 24hr >90% >90% 65% 60%
COS7 % RLU Max (ratio; vol.) MAX (3:1; 10µl) 90%(3:1; 5µl) 80% (4:1; 10µl) 90% (4:1; 10µl)
% Viable @ 24hr >90% >90% >90% 85%
H9C2 % RLU Max (ratio; vol.) 10% <10% MAX (4:1; 10µl) 50% (3:1; 10µl)
% Viable @ 24hr >90% >90% >90% 85%
HCT116 % RLU Max (ratio; vol.) 15% MAX (2.5:1; 5µl) 95% (2:1; 5µl) 60% (2:1; 5µl)
% Viable @ 24hr 80->90% 70% 80% 70%
HEK 293 % RLU Max (ratio; vol.) 60% (4:1; 10µl) 20% MAX (4:1; 10µl) 30%
% Viable @ 24hr 75% 60% >90% 75%
HeLa % RLU Max (ratio; vol.) 30% MAX (3:1; 5µl) 60% (4:1; 10µl) 45%
% Viable @ 24hr 80% 80% >85% 70%
HepG2 % RLU Max (ratio; vol.) 30% 30% MAX (4:1; 10µl) 30%
% Viable @ 24hr 75% 55% 55% 65%
HT-29 % RLU Max (ratio; vol.) <10% 40% MAX (3:1; 10µl) 90% (3:1; 10µl)
% Viable @ 24hr >90% >90% >90% >90%
Huh7 % RLU Max (ratio; vol.) 40% 10% MAX (2.5:1; 5µl) 60% (2:1; 5µl)
% Viable @ 24hr 80% 75% 75% 70%
Jurkat % RLU Max (ratio; vol.) <10% <10% MAX (2:1; 5µl) 65% (2:1; 10µl)
% Viable @ 24hr >90% >90% >90% 80%
K562 % RLU Max (ratio; vol.) <10% 10% 30% MAX (2:1; 10µl)
% Viable @ 24hr >90% >90% >90% >90%
LNCaP % RLU Max (ratio; vol.) <10% 40% MAX (4:1; 10µl) 20%
% Viable @ 24hr >90% 75% >90% 50%
MCF7 % RLU Max (ratio; vol.) 10% 15% 80% (4:1; 10µl) MAX (2:1; 10µl)
% Viable @ 24hr >90% >90% >90% 80%
NIH 3T3 % RLU Max (ratio; vol.) <10% 15% 60% (4:1; 10µl) MAX (4:1; 10µl)
% Viable @ 24hr >90% >90% >90% >90%
PC-12 % RLU Max (ratio; vol.) 10% 70% (2.5: 1; 10µl) MAX (4:1; 10µl) 90% (2:1; 10µl)
% Viable @ 24hr >90% >90% >90% >90%
PC-3 % RLU Max (ratio; vol.) 15% 90% (4:1; 10µl) MAX (4:1; 10µl) 15%
% Viable @ 24hr >90% >90% 80% 40-70%
RAW 264.7 % RLU Max (ratio; vol.) MAX (3:1; 10µl) 85% (2.5:1; 5µl) <10% 50% (2:1; 5µl)
% Viable @ 24hr 75% 65% >90% 70%
U2OS % RLU Max (ratio; vol.) 40% 60% (3:1; 5µl) MAX (4:1; 10µl) 20%
% Viable @ 24hr >90% >90% >90% >90%
+++ ≥80% of RLU max; viability >80% at 24hr. ++ 50% to <80% RLU max; viability >50% to <80% at 24hr.
L2K, Lipofectamine® 2000.

For most lipid-based reagents, manufacturers recommend starting with a reagent:DNA ratio of 3:1, using 100ng of DNA per well in a 96-well plate. Table 3 compares transfection reagents using these conditions for the cell lines tested, using data extracted from the overall transfection study. Luciferase expression (EXP) was determined for each cell line relative to the maximum RLUs observed (MAX). Viability (VIA) was measured as a percentage of untransfected cells for each cell line. Under the tested conditions, ViaFect™ Reagent offered the best combination of transfection efficiency and low toxicity for most cell lines, making it an ideal choice when beginning transfection experiments with a new cell line.

Table 3. Reagent comparison under standard recommended conditions.

Cell Line FuGENE® 6 FuGENE® HD ViaFect™ L2K
  EXP VIA EXP VIA EXP VIA EXP VIA
A549 60% >90% 60% 60% MAX >90% <10% 30%
C2C12 15% >90% <10% >90% MAX >90% 15% >90%
CHO 55% >90% 40% >90% MAX 85% 50% 45%
COS7 MAX 85% 90% >90% 60% >90% 85% >90%
H9C2 15% >90% <10% >90% MAX >90% 65% >90%
HCT116 20% 80% 80% 65% MAX 80% 40% 45%
HEK 293 70% 75% 20% 50% MAX >90% 30% 40%
HeLa 25% >90% MAX 65% 65 >90% 40% 50%
HepG2 35% 85% 20% 50% MAX 55% 25% 55%
HT-29 <10% >90% 45% >90% MAX >90% 90% 85%
Huh7 40% 70% <10% 40% MAX 70% 25% 45%
Jurkat 35% >90% <10% >90% MAX 80% 95% 80%
K562 <10% >90% <10% >90% 20% >90% MAX >90%
LNCaP <10% >90% 60% 75% MAX >90% 25% 60%
MCF7 <10% >90% 20% >90% 95% >90% MAX 70%
NIH 3T3 <10% >90% 25% >90% 65% >90% MAX >90%
PC-12 15% >90% 70% >90% MAX >90% 95% 85%
PC-3 20% >90% 55% 80% MAX >90% 15% 65%
RAW 264.7 MAX 75% 45% 55% <10% >90% 35% 45%
U2OS 40% >90% 70% >90% MAX >90% 25% >90%
+++ ≥80% of RLU max ++ >50% to <80% of RLU max
L2K, Lipofectamine® 2000

General Guidelines for Transfection Optimization

With any transfection reagent, conditions will need to be optimized for best results. The following guidelines are applicable to transfection of mammalian cell lines.

Transfect healthy, actively dividing cells at a consistent cell density. Cells should be at a low passage number and 50–80% confluent when transfected. Using the same cell density reduces variability for replicates. Ensure that cells are free of Mycoplasma contamination for optimal growth.

Use high-quality DNA. Transfection-quality DNA is free from protein, RNA and chemical contamination with an A260/A280 ratio of 1.7–1.9. Prepare purified DNA in sterile water or TE buffer at a final concentration of 0.2–1mg/ml.

Optimize the amount of DNA used to transfect cells. The optimal amount of DNA to use in the transfection will vary widely, depending upon the type of DNA and target cell line used. We recommend initially testing 50–200ng of DNA per well in a 96-well plate format. As shown in the experiments reported here, increasing the amount of DNA does not necessarily result in higher transfection efficiencies.

Optimize the transfection reagent:DNA ratio. For many cell lines, ratios of 1.5:1–4:1 (microliters reagent to micrograms DNA) work well (see Table 2), but ratios outside this range may be optimal for a particular cell type or application. Test other ratios to maximize the transfection efficiency. The FuGENE® HD Protocol Database provides optimization guidelines for a variety of cell lines.

Determine the best reporter gene for measuring transfection efficiency. The experiments reported here use firefly luciferase as a reporter gene, but other reporters can be used. An ideal reporter gene product is unique to the cell, can be expressed from plasmid DNA and can be assayed conveniently. Generally, such assays are performed 24–48 hours after transfection.

Optimize cell density. The plating density for a specific cell line will depend on the growth rate. A general guideline is to plate ~5 × 105 adherent cells per 60mm culture dish or 1 × 106 suspension cells per assay. For 96-well plates, we generally recommend 1–2 × 104 adherent cells or 2–10 × 105 suspension cells per well. Scale the number of cells proportionally if using different size plates.

Determine reagent compatibility with serum. Many transfection protocols require serum-free conditions for optimal performance because serum can interfere with some commercially available transfection reagents. ViaFect™ Transfection Reagent can be used in the presence of serum for transfecting cell types that require continuous exposure to serum, such as primary cell cultures.

Incubate DNA with transfection reagent for the optimal time. Transfection reagents need time to form the transfection reagent:DNA complex (e.g., 5–20 minutes at room temperature for the ViaFect™ Transfection Reagent). The optimal time for complex formation varies for each cell line. Incubate transfected cells for 24–48 hours before assaying to allow time for expression of transfected DNA. 

 

Conclusion

All the transfection reagents tested work on a variety of cells. Each reagent performs best with certain cell lines, but no one reagent was perfect for every cell line. The commonly used 3:1 reagent:DNA ratio is not always the most optimal condition. In this study, ViaFect™ Reagent yielded greater luciferase expression over a wider variety of cells, under optimized or standard transfection conditions, compared to other reagents tested.

 

How to Cite This Article

Hook, B. and Landreman, A. Choosing the Right Transfection Reagent for Optimal Efficiency. [Internet] November 2018. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/tpub-205-choosing-the-right-transfection-reagent-for-optimal-efficiency/

Hook, B. and Landreman, A. Choosing the Right Transfection Reagent for Optimal Efficiency. Promega Corporation Web site. http://www.promega.com/resources/pubhub/tpub-205-choosing-the-right-transfection-reagent-for-optimal-efficiency/ Updated November 2018. Accessed Month Day, Year.

CellTiter-Fluor, ONE-Glo and ViaFect are trademarks of Promega Corporation.

FuGENE is a registered trademark of Fugent, LLC. Lipofectamine is a registered trademark of Thermo Fisher Scientific.