What transfection reagent should I choose?
Promega offers three premium transfection reagents:
ViaFect™, FuGENE® 6 and FuGENE® HD. All threeoffer broad spectrum transfection capability and low
toxicity but the same question always comes up:
Which one should I choose? First, not everytransfection reagent is perfect for every cell line.
Second, transfection success can also vary with DNA
quality, passage number of cells, cell density, etc.
To get a better understanding of how each reagent
performs on a variety of cell lines, a transfection trial
was performed with the three Promega reagents andthe leading competitor’s transfection reagent . Eachreagent was tested with a range of transfection
reagent: DNA ratios and two concentrations of DNA.

Experiments
See page 2. Cells were transfected with either 50ng
or 100ng of luciferase reporter plasmid at various
reagent:DNA ratios.

Table 1. Summary of Transfection Study.
Transfection was assessed through expression of firefly
luciferase (EXP). The best condition (highest RLUs) waschosen for each transfection reagent. For each cell line, themaximum RLUs were set at 100% for one reagent and theother 3 were judged relative to that reading. Cells were also
assayed for cell viability (VIA) and judged versus a notransfection control for each cell line. Transfection
efficiencies lower than 50% are not reported on this table.
Greater detail is reported in Table 2.
CellLineA549

FuGENE® 6

A summary of the results are presented in Table 1
with more information on the exact best conditions inTable 2.
The leading competitor and the Promega reagentssuggest trying a 3:1 ratio with 100ng of DNA per wellof a 96-well plate if you need a starting place. Table 3demonstrates how the reagents perform relative to
one another when you use the common conditions.
.

Conclusion
All four transfection reagents work on a variety ofcells. Each reagent clearly works best on certain celllines but none were perfect for every cell line. Table 2
demonstrates that the common 3:1 ratio is not always
the most optimal condition. ViaFect™ Reagent yielded
greater luciferase expression over a wider variety ofcells in this test whether you optimize or just choose
one condition.

VIA++++ +++ +++ +++ +++

VIA

EXP

VIA

Competitor
L2KEXPVIA+++++++ +++

C2C12CHO++ +++

COS7+++ +++ +++ +++ +++ +++ +++ +++H9C2+++ +++ ++ +++HEK 293+++ +++++ ++ +++ +++ +++++++++++ ++++++ +++ ++ +++HeLa
HepG2+++ ++HT-29+++ +++ +++ +++Huh7+++ ++Jurkat+++ +++ ++ +++K562+++ +++++

++

LNCaP

+++ +++

MCF7+++ +++ +++ +++NIH 3T3++ +++ +++ +++PC-12++ +++ +++ +++ +++ +++PC-3+++ +++ +++ +++RAW264.7+++ ++ +++ ++• >80% of maxRLUs;

KEY++

++

++ +++ +++ +++U2OS

This summary is based on data generated by
Brad Hook and Alisha Truman, Promega Scientific
Applications Services, June 2014.

EXP

ViaFect™
Reagent

EXP

HCT116

Results

FuGENE®
HD+++ • >80% of cells wereviable• >50% to <80% max RLUs;++• >50% to <80% of cells wereviable1Experimental Overview
1. Transfection reagents were prepared at room
temperature following the directions for eachtransfection reagents. The following reagent toDNA ratios were tested:
FuGENE® 6Reagent

6:1

4:1

3:1

1.5:1

FuGENE® HDReagent

4:1

3:1

2.5:1

2:1

ViaFect™
Reagent

4:1

3:1

2.5:1

2:1

Competitor
L2K

5:1

4:1

3:1

2:1

2. Each reagent:DNA ratio was pipetted onto 6wells of cells in either 5µl or 10µl volumes. One
96 well plate/transfection reagent was used percell line.
3. Plates were mixed for 15 seconds on a plateshaker for 10-15 seconds
4. Plates were transferred to a 37°C/5% CO2
incubator for 24 hours prior to assay for cell
viability and luciferase reporter activity. Wells
with cells only were 100% viability controls.
More complete experimental details are available
upon request.

Example datawith HeLa cells

2Table 2. Optimal 96-well transfection conditions for each cell line at
24 hours post-transfection.
Transfection was judged through expression of firefly luciferase. The conditions that produced thehighest RLUs for each transfection reagent were compared relative to the reagent that produced thehighest 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 thatreagent. Viability was judged relative to untransfected control cells. Details for luciferase expression
below 50% of MAX RLUs are not reported in this table.
Cell Line
A549

MeasureFuGENE® 6FuGENE® HD

ViaFect™

L2K% RLU Max (ratio; vol.)60% (4:1; 10µl)

90% (3:1; 5µl)

MAX (4:1; 10µl)

30%% Viable @ 24hr

>90%>90%>90%

60%% RLU Max (ratio; vol.)<10%<10%MAX (4:1; 10µl)

25%% Viable @ 24hr

>90%>90%>90%>90%

60% (6:1; 5µl)

30%MAX (4:1; 10µl)

65% (2:1; 10µl)

C2C12CHO
COS7
H9C2
HCT116
HEK 293
HeLa
HepG2HT-29
Huh7
Jurkat
K562
LNCaP
MCF7
NIH 3T3
PC-12PC-3
RAW 264.7
U2OS% RLU Max (ratio; vol.)% Viable @ 24hr

>90%>90%

65%

60%

MAX (3:1; 10µl)

90%(3:1; 5µl)

80% (4:1; 10µl)

90% (4:1; 10µl)% Viable @ 24hr

>90%>90%>90%

85%% RLU Max (ratio; vol.)10%<10%MAX (4:1; 10µl)

50% (3:1; 10µl)% Viable @ 24hr

>90%>90%>90%

85%% RLU Max (ratio; vol.)% RLU Max (ratio; vol.)15%MAX (2.5:1; 5µl)

95% (2:1; 5µl)

60% (2:1; 5µl)

80->90%

70%

80%

70%

60% (4:1; 10µl)

20%

MAX (4:1; 10µl)

30%% Viable @ 24hr

75%60%>90%75%% RLU Max (ratio; vol.)30%

MAX (3:1; 5µl)

60% (4:1; 10µl)

45%% Viable @ 24hr

80%

80%

>85%

70%

% RLU Max (ratio; vol.)

30%

30%

MAX (4:1; 10µl)

30%% Viable @ 24hr

75%

55%

55%

65%

% RLU Max (ratio; vol.)<10%40%MAX (3:1; 10µl)

90% (3:1; 10µl)% Viable @ 24hr
% RLU Max (ratio; vol.)% Viable @ 24hr

>90%>90%>90%>90%% RLU Max (ratio; vol.)40%

10%MAX (2.5:1; 5µl)

60% (2:1; 5µl)

% Viable @ 24hr

80%75%

75%

70%% RLU Max (ratio; vol.)<10%<10%MAX (2:1; 5µl)

65% (2:1; 10µl)% Viable @ 24hr

>90%>90%>90%

80%% RLU Max (ratio; vol.)

<10%

10%

30%MAX (2:1; 10µl)% Viable @ 24hr

>90%>90%>90%>90%% RLU Max (ratio; vol.)<10%40%MAX (4:1; 10µl)

20%% Viable @ 24hr

>90%75%>90%

50%% RLU Max (ratio; vol.)10%

15%80% (4:1; 10µl)

MAX (2:1; 10µl)% Viable @ 24hr

>90%>90%>90%

80%% RLU Max (ratio; vol.)<10%15%60% (4:1; 10µl)

MAX (4:1; 10µl)% Viable @ 24hr

>90%>90%>90%>90%% RLU Max (ratio; vol.)10%

70% (2.5: 1; 10µl)

MAX (4:1; 10µl)

90% (2:1; 10µl)% Viable @ 24hr

>90%>90%>90%>90%% RLU Max (ratio; vol.)15%

90% (4:1; 10µl)

MAX (4:1; 10µl)

15%% Viable @ 24hr

>90%>90%80%

40-70%MAX (3:1; 10µl)

85% (2.5:1; 5µl)

<10%50% (2:1; 5µl)

% RLU Max (ratio; vol.)% Viable @ 24hr

75%

65%>90%70%% 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% @ 24hr

50 to <80% of RLU Max>50% to <80% Viability @ 24hr

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Table 3: Comparison of all four transfection reagents at the commonly
recommended conditions of 3:1 Reagent:DNA ratio and 100ng DNA per
well of a 96-well plate.
This data was extracted from the overall transfection study. Luciferase expression (EXP)
is judged for each cell line relative to the maximum RLUs observed (MAX). Viability (VIA;% of untransfected cells) is listed for each cell line under these conditions.
FuGENE® 6

FuGENE®
HD

ViaFect™
Reagent

Competitor
L2K

EXP

VIA

EXP

VIA

EXP

VIA

EXP

VIA

A549

60%>90%

60%

60%

MAX

>90%

<10%30%C2C1215%>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%

Jurkat35%>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-1215%>90%70%>90%

MAX>90%95%85%

PC-320%>90%

55%

80%

MAX>90%15%

65%

RAW 264.7

MAX75%

45%

55%<10%>90%35%

45%

U2OS

40%>90%70%>90%

MAX>90%25%>90%KEY≥80% MAX RLUs

Cell Line

Product
ViaFect™ Transfection Reagent

FuGENE® HD Transfection Reagent

FuGENE® 6 Transfection Reagent>50% to <80% MAX RLUs

SizeCat. #0.75ml

E4981

2 x 0.75ml

E4982

1ml

E2311

5 x 1ml

E2312

1ml

E2691

5 x 1ml

E2692

CellTiter-Fluor, ONE-Glo and ViaFect aretrademarks of Promega
Corporation. Products may be covered by issued or pending patents
or subject to use limitations, please visit www.promega.com for more
information.
FuGENE is a registered trademark of Fugent, L.L.C.

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