Luciferase Assay System
TECHNICAL NOTE:

Co-transfectioncontrols with reporter assays
TECHNICAL NOTE:
Co-transfection controls with reporter assays

Luciferase Assay System

Do I need a co-transfection control?
Do I need a co-transfection control?
In most cases you do not need a co-transfection control. This technical bulletin offers guidelines andexperimental data that will help you evaluate the needs of your reporter assay experiment.
In most cases you do not need a co-transfection control. This technical bulletin offers guidelines and experimental data that willhelp you evaluate the needs of your reporter assay experiment.

What does a co-transfection control do?
What doesa co-transfection
control withdo?a reference or control plasmid to help normalize
Historically,an experimental
plasmid was co-transfected
two main sources of variation between transfection replicates:
Historically, an experimental plasmid was co-transfected with a reference or control plasmid to help normalize two main sources
1. between
Low andvariable replicates:transfection efficiencyof variationtransfection
2.
Variablecelllysis
and variable
lysate stability
1. Low and variable transfection
efficiency
2. Variable cell lysis and variable lysate stability
In the days of calcium phosphate transfections and home brew assay reagents, co-transfections wereessential.
ultra
high-efficiency
transfection
reagents
such as FuGENE
and optimizedlysis
In the daysHowever,of calcium modern
phosphatetransfections
and home
brew assay reagents,
co-transfections
wereHDessential.
However, modern
bufferandassay
reagents
suchastheLightSwitch
Assay
Reagent
often
reducethesetypesofvariation
toultra high-efficiency transfection reagents such as FuGENE HD and optimized lysis buffer and assay reagents such as the Lightcoefficientsvariance
less types
thanof10%(Table
Switch Assay ofReagent
often(CVs)reduceof
thesevariation
to1).
coefficients of variance (CVs) of less than 10% (Table 1).
Co-transfection
controls
wereoriginallydesigned
to removevariability
due to transfection
alone(1). were
Co-transfection controls
wereoriginallydesigned
to removevariability
due to transfection
efficiency efficiency
alone (1). Suchcontrols
Suchcontrols
werenotdesignednoraretheyadequate
forcomparing
betweendifferent
conditions
orcelllines.
not designed nor are they adequate for comparing between different conditions or cell lines.
Promoter (pGL4 vector)%CV Single Tfx(3 reps)%CV Co-Tfx normalized(3 reps)

TKACTB

RPL10 ANKRD37 EGLN1

ENO2

AVG

14%

22%

11%

7%

12%

18%

2%

13%

8%

12%

6%

4%

7%

9%

Table 1: Variation between replicate transfections is the same for single transfections and normalized co-transfections
InTablethe singletransfection(tfx) experiment,
6 different
promoter-luc2P
constructs
independently
1: Variation
betweenreplicatetransfections
is the reportersame forsingle weretransfections
andtransfected
into HepG2 cells without a co-transfection control in triplicate and assayed with Steady-Glo (Promega) 24 hrs after
normalized co-transfectionstransfection. Additionally, the same 6 promoter-luc2P vectors were co-transfected with a TK-renilla plasmid and assayed
In the single transfection (tfx) experiment, 6 different promoter-luc2P reporter constructs were independently transfected intowith Dual-Glo (Promega). The co-transfected plasmid data were normalized by dividing the firefly signal by the renilla
HepG2Thecellswithouta co-transfection
control inoftriplicateandassayedwith Steady-Glo(Promega)24 hrs
after transfection.
signal.
tableaboveshows the coefficientvariation(%CV=stdev/mean)of the3 replicatesof eachconstruct. The
Additionally,
thesame6
promoter-luc2P
vectors
wereco-transfectedwithaTK-renilla
plasmidandassayedwith Dual-Glo
average %CV for the single transfection is 9%, whereas the average %CV for the normalized co-transfection
is 12%.

(Promega). The co-transfected plasmid data were normalized by dividing the firefly signal by the renilla signal. The table aboveshows the coefficient of variation (%CV= stdev/mean) of the 3 replicates of each construct. The average %CV for the single
transfection is 9%, whereas the average %CV for the normalized co-transfection is 12%.

Are there any down-sides to doing a co-transfection control?
Co-transfecting two plasmids is significantly different than transfecting a single plasmid. The effect of aco-transfection in your experimental design should be carefully considered. The most important considerations
should be the following:
Co-transfecting two plasmids is significantly different than transfecting a single plasmid. The effect of a co-transfection in yourshould becarefully considered.
The most importantconsiderations
shouldbe the following:
1.experimental
Time, Cost,design
and Signal:
Dual-reporter
assay reagentsare morethan twice as
expensive
and take more than

Are there any down-sides to doing a co-transfection control?

2 times as long as a single transfection. A single transfection design can double the scope of a project that
1. Time,Cost, andinSignal:
assay reagentsare more thanassay
twice asexpensiveyieldand takemore
2 timesascanbe completed
half of Dual-reporterthe time. Furthermore,dual-reporter
reagents
lowerrawthan
signalthanlong as a singletransfection.
A single transfection
designdoublereporter
the scopeproteinof a projectthat 1).
can be completed in half of thereagentsthat havebeen optimized
specificallyforcana single(Figuretime. Furthermore, dual-reporter assay reagents yield lower raw signal than reagents that have been optimized specifically for asingle reporter protein (Figure 1).
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Luciferase Assay System

2. Interaction between vectors: Co-transfected plasmids have long been known to interact with each other.
Strong promoters can sequester basal factors and artificially repress the signal of the control. Conversely,
promoters with inducible response elements can trans-activate the promoter on the control vector. Sorting out
these confounding effects can be extremely challenging and give results that are difficult to interpret.
3. Reliance on a single control promoter: Dual-reporter assay reagents are more than twice as expensive andtake more than 2 times as long as a single transfection. A single transfection design can double the scope ofa project that can be completed in half of the time. Furthermore, dual-reporter assay reagents yield lower raw
signal than reagents that have been optimized specifically for a single reporter protein (Figure 1).

Figure 1: Relative luminescence from single and co-transfected promoter reporter vectors
In the single transfection (tfx) experiment, 6 different promoter-luc2P reporter constructs were independently transfected
into HepG2 cells without a co-transfection control in triplicate and assayed with Steady-Glo (Promega) 24 hrs aftertransfection. Additionally, the same 6 promoter-luc2P vectors were co-transfected with a TK-renilla plasmid and assayedwith Dual-Glo (Promega). The data from both experiments represent raw (un-normalized) firefly luminescence. Theaverage RLU and standard deviation of each construct is shown in the bar chart above.

If I don’t use a co-transfection control, which controls should I use?
One of the big advantages of using a single transfection design is that you can include many different
control vectors in your experiment, such as various housekeeping promoters, to allow comparisons acrossa variety of cell lines or treatment conditions. In this experimental setup, instead of one control construct
being co-transfected with every experimental plasmid, many different control promoters are included in theexperiment, each in its own well, in addition to the experimental promoters to be tested. Because no single
promoter will serve as an ideal control, LightSwitch offers a panel of positive and negative control vectors tomeasure background and non-specific responses with the highest degree of confidence.

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Luciferase Assay System

How do I decide whether I need a co-transfection control?
The easiest thing to do is to try your experiment without co-transfecting the control plasmid and measure
the variation between replicate single transfections. More often than not, your %CV will be within an
acceptable limit (Table 1). With few exceptions, your experimental conclusions will be the same as if you useda co-transfection control (Figure 2). If you feel like your experiment does require a co-transfection control, theLightSwitch Dual Assay System is fully compatible and optimized for use with LightSwitch GoClones.

Figure 2: Hypoxia promoter induction ratios are the same with and without a co-transfection control
In this experiment, two housekeeping control promoters and two hypoxia-inducible promoters were transfected in triplicate
with and without a co-transfection control. The 4 promoters were exposed to either 100uM DFO or normal media for 24 hrs
and luciferase activity was measured with Steady-Glo (single transfection) or Dual-Glo (co-transfection). The graph aboveshows the log2 ratio of the DFO treated to untreated activity for each of the 4 promoters. The fold induction for eachpromoter is not significantly different between the single and co-transfected vectors.

References
1. Schagat, T., Paguio, A., and Kopish, K. 1996. Normalizing genetic reporter assays: approaches andconsiderations for increasing consistency and statistical significance. Cell Notes 17:9-12.
2. Shifera, A., and Hardin, J. 2010. Factors modulating expression of Renilla luciferase from control plasmids
used in luciferase reporter gene assays. Analytical Biochemistry. 396:167-172.
3. Ho, C., and Strauss, J. 2004. Activation of the control reporter plasmids pRL-TK and pRL-SV40 by multiple GATA
transcription factors can lead to aberrant normalization of transfection efficiency. BMC Biotechnology. 4:10.
4. Matuszyk, J., Ziolo, E., Malgorzata, C., Kochel, I., and Strzadala, L. 2002. Nurr1 affects pRL-TK but not phRG-B
internal control plasmid in genetic reporter system. Biochem. Biophys. Res. Comm. 294: 1036-1039.

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