Reporter Constructs, Assays & Services

LightSwitch™ Luciferase Assay System
Promoter Reporter GoClones™
Synthetic Response Elements
Pathway Reporter Cell Lines
3´UTR Reporter GoClones™
miRNA Mimics & Inhibitors
Synthetic miRNA Targets
Transfection Reagents

Gene expression is controlled at both the transcriptional and posttranscriptional levels. Transcription factors and other DNA-binding
proteins are important regulators of transcription initiation, while
microRNAs and the RISC complex interact with the 3´UTR of genetranscripts to control mRNA stability and translation.
The LightSwitch™ Luciferase Assay System is a comprehensive
system for studying transcriptional and post-transcriptional
regulation. It includes over 30,000 pre-cloned promoter and3´UTR reporter constructs, optimized companion reagents, andfull protocols that support your project from start to finish.

Luciferase Assay Kits
Custom Services
Enabling Epigenetics ResearchLightSwitch™ – comprehensive, optimized luciferase reporter system
The LightSwitch™ Luciferase Assay System is a complete solution for studying transcriptional & post-transcriptional
gene regulation in living mammalian cells by directly measuring the functional activity of promoters and 3´UTRs.
With GoClone™ Collections of over 18,000 human promoters and 12,000 3´UTRs available as transfection-readyLightSwitch luciferase reporter vectors, you can perform your reporter assay experiments immediately, without theneed to clone or prepare DNA.
The LightSwitch System utilizes an engineered luciferase gene (RenSP) and optimized assay & transfection reagentsthat are designed to provide superior results with easy-to-use protocols. Other products in the system include
validated positive and negative control vectors, stable reporter cell lines, miRNA mimics & inhibitors, and collectionsof synthetic long-range response element constructs and synthetic 3´UTR target validation constructs. We also offer
comprehensive services for custom cloning, mutagenesis, pathway screening, miRNA target validation and sequencevariant analysis. For more information, please visit www.activemotif.com/lightswitch.
Advantages of the LightSwitch Luciferase Assay System• Fast & convenient – LightSwitch GoClones aresequence-verified, transfection-ready humanpromoter and 3´UTR reporter constructs. No
cloning, DNA preparation or assay optimization is needed. To begin, simply search foryour regulatory elements of interest using
our online search tool (Figure 1).
• Enhanced sensitivity – We also offer collections of cloned synthetic regulatory elementsthat can provide stronger responses than theendogenous promoter and 3´UTR sequences.

Search for Promoter & 3´UTR Reporter constructs at www.activemotif.com/goclones.

SEARCH BY GENE IDENTIFIER
In the box below, type or paste in a list of one or more gene identifierson separate lines. Click on one of the buttons below to search.
DNMT3ADNMT3B
Search for:Promoter

3´UTR

Both

• Quantitative – All LightSwitch vectors contain
RenSP, an optimized luciferase gene that
our scientists have engineered for increased
brightness and responsiveness (Back Cover).
Our LightSwitch Luciferase Assay Substrate
was formulated specifically for use with
RenSP to provide high sensitivity over abroad dynamic range.

Prod ID

Gene Symbol

Type

Sequence of Cloned Elements

Format

S709833

DNMT3APROMOTER

Sequence and Clone Info

5 µg

S709808

DNMT3B

PROMOTER

Sequence and Clone Info

5 µg

• Cost-effective – The LightSwitch Assay scales
up easily, making it possible to efficiently
screen large numbers of clones for activation
and/or repression under a multitude ofdifferent conditions.

Figure 1: Online search tool for finding pre-cloned LightSwitch GoClone Promoter and 3´UTR constructs.
Finding cloned regulatory elements for your genes of interest is fast and easy. Simply enter your gene’s name,accession number or other identifier in the search box and click a button to search for promoter constructs,
3´UTR constructs, or both. The search results detail which GoClone constructs are available for your elements,
including links to view the cloned sequence as well as gene information at NCBI. In this example, a search was
performed for both promoter and 3´UTR constructs of DNMT3A and DNMT3B.

S808608

DNMT3A

3´UTRSequence and Clone Info

5 µg

S809202

DNMT3B

3´UTRSequence and Clone Info

5 µg

www.activemotif.com

Selecting LightSwitch Luciferase Assay System products for your experiments
Step 1: Select or prepare your reporter constructs
Choose from our collection of over 30,000
human promoters and 3´UTRs (Figure 1), TF
response elements, synthetic miRNA target
reporters, or clone your own promoter,3´UTR or enhancer elements into the appropriate empty LightSwitch Reporter vector.
Alternatively, our Custom Services team canquickly and economically clone any fragmentfrom the human, mouse or rat genome into
any LightSwitch vector for you.

Human or other mammalian genomes
EnhancerCloned into
Reporter
Vector//

Gene of interest

PromoterCloned into
Reporter
RenSP
VectorLuciferase

LightSwitch™
LR Enhancer
Reporter

RenSP
Luciferase

LightSwitch™
Promoter
ReporterStep 2: Choose appropriate positive &negative control constructs

Step 3: Select miRNA mimics and inhibitors(for 3´UTR-miRNA experiments)

Because no single promoter can serve as an ideal
control for all experimental conditions, we offer
a panel of positive and negative controls for use
with LightSwitch Promoter & 3´UTR Reporter vectors. These control constructs are used to assesstransfection efficiency, measure background, and tonormalize for non-specific effects associated with atreatment or change in condition.

When assessing the functional
impact of miRNA-3´UTR interactions, you will need to selectmiRNA mimics and/or inhibitors,
which will be co-transfectedalong with your 3´UTR Reporter
or Synthetic miRNA Target
Reporter constructs.

5´

3´UTR

Cloned into
Reporter
RenSP
Luciferase Vector

LightSwitch™
3´UTR
Reporter

3´UTR
mRNA

RISC

AAA-3´miRNA

Step 4: Choose transfection reagents
For conducting plasmid transfections andco-transfections, we offer FuGENE® HD
Transfection Reagent because of its
superior efficiency and low cytotoxicity
across a wide variety of cell lines. If you areco-transfecting plasmids and miRNA mimics
or inhibitors, we offer DharmaFECT® Duo
Transfection Reagent, which we have foundto be the most efficient reagent forco-transfecting plasmids and small RNAs.

Step 5: Select a LightSwitch Luciferase Assay or Dual Assay Kit
All LightSwitch reporter constructs containthe optimized RenSP luciferase gene, andLightSwitch Luciferase Assay Reagents MUST
be used to obtain optimal results. The kit
contains a novel, proprietary substrate thatwas formulated specifically for use with
RenSP. (Other luciferase assay reagents are
not compatible with RenSP.) The kit’s assay
buffer was designed to enable one-stepreagent addition, eliminating the need toperform a separate lysis step.

LightSwitch
Reporter
GoCloneLight
Signal 1
RenSP
Luciferase

Cypridina
Luciferase
Control

Light
Signal 2
Cypridina
Luciferase

Due to the availability of improved
transfection and assay reagents, there istypically no longer a need to co-transfecta control plasmid to normalize luciferase
assays (Table 1, Back Cover). However, fordifficult-to-transfect cell lines, we offer theLightSwitch Dual Assay Kit, which is used toassay both RenSP and Cypridina luciferases.
Note, positive & negative control constructs
should still be used to normalize between
your experimental conditions.

For more complete information on all LightSwitch products, please visit www.activemotif.com/lightswitch.

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LightSwitch™ – comprehensive, optimized luciferase reporter system
Study regulation of promoter activity modulated by transcription factor function
Transcription factors are important regulatorsof eukaryotic transcription. They interact
with DNA-encoded regulatory elements likepromoters to modulate when, where andhow much of a gene’s mRNA is produced.
The activity of transcription factors can beaffected by the cell’s environmental conditions including exposure to signaling molecules like hormones and growth factors.

Human genome

Reporter assays are valuable for directly
measuring promoter activity in living cells.
They are a simple and scalable solution formonitoring changes in the transcriptional
activity of a promoter. Changes in activity
may be induced by over-expression of acDNA, knockdown by an siRNA or treatment
with a small molecule.

Promoter

Gene of interest

Pre-cloned intoa Reporter
VectorRenSP
Luciferase

LightSwitch™
Promoter
Reporter
1. Transfectvector.

Applications for LightSwitch promoter products• Monitor promoter activity in response to variations in transcription factor function.

2. Treat withcompounds,
pathogens,or altered
growthconditions.

• Understand the mechanisms by which transcription is induced or repressed.
• Assess the functional consequences of binding events detected through ChIP.
• Dissect transcription factor motif function using mutagenesis.
• Measure the effects of naturally occurring sequence variants.
Light signal• Generate dose response curves to various test compounds or growth conditions.

LightSwitch promoter product portfolio
The LightSwitch Luciferase Assay System makes it fast & easy to measure the functional effectsof transcription factor activity. After a promoter of interest is cloned upstream of RenSP in theLightSwitch Promoter Reporter vector, it is transfected into live cells. Any changes in promoter
activity cause changes in the reporter signal, which is measured as light output (Figures 2 & 3).

• LightSwitch Synthetic Response Elements – These constructs include multiple repeats ofa transcription factor binding site motif cloned upstream of a minimal promoter and RenSP.
In some cases these response elements provide higher sensitivity than the endogenous
human promoters found in the Promoter GoClone Collection, making them well suited foruse in primary screens to identify compounds or conditions that affect biological pathways.
• LightSwitch Pathway Reporter Stable Cell Lines – These cell lines contain a stably integrated, human regulatory element driving expression of RenSP. Each has been validated
on several plate-based formats for measuring the activity of a specific biological pathway,
making them ideal for both primary and secondary high-throughput screening.
• Validated Promoter Controls – Constructs for use as positive & negative controls.
• Empty Promoter & Long-range Enhancer reporter vectors – Clone your own sequences.
• Pathway Screening Services to study disease-related biological pathways (see other side)
For more complete information, please visit www.activemotif.com/ls-promoter.

Figure 2: LightSwitch Promoter Reporter Assays.
To study a promoter’s regulation, it is cloned into aLightSwitch Promoter Reporter vector and transfected.
The cells are treated, if desired, and then assayed. The
luciferase produced oxidizes the assay substrate in areaction that also produces light. The amount of lightis proportional to the effect of the cloned promoter.

Relative Luminescence Units• LightSwitch Promoter Reporter GoClone Collection – An algorithm was used to evaluate
over 5 million human cDNA sequences to identify transcription start sites (TSS) throughout
the human genome. Based on these TSS predictions, over 18,000 human promoter sequences of ~1 kb (–900…+100 bp relative to the TSS) were cloned into the LightSwitch Promoter
Reporter vector. Each GoClone is purified and sequence-verified during our extensive
quality control process, and is delivered ready for transfection. Simply find and order yourreporter clones using our online search tool (Figure 1), and then begin your assays.

3. Measure luciferase activity to determine
the functional effects of TF binding,treatments and/or sequence variations.

400,000
300,000
200,000
100,000
0
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-11

-10

-9

M (log10)-8-7

Figure 3: Dose response of LightSwitch SYT8 Promoter
Reporter construct to various estrogen compounds.
HT1080 cells were co-transfected with a SYT8 Promoter
GoClone (Product ID S714388) and an ER cDNA
expression plasmid, then treated with five estrogencompounds for 24 hours before the luminescence wasmeasured using the LightSwitch Luciferase Assay Kit.

www.activemotif.com

Study post-transcriptional regulation mediated through miRNA-3´UTR interactions
MicroRNAs (miRNAs) are small, non-coding
RNA molecules of ~22 nucleotides that playa large role as post-transcriptional regulators.
miRNAs are thought to repress expression
by targeting the 3´UTR (untranslated) regionof mRNA transcripts. The interaction of amiRNA with a 3´UTR results in either
inhibition of translation or increased
degradation of the targeted transcript.

Human genome

While the human genome is thought toencode only about 1000 miRNAs, they are
believed to target ~60% of mRNA transcripts.
A given miRNA may have multiple 3´UTRtargets, and a given 3´UTR may be targetedby multiple miRNAs. Therefore, there isconsiderable interest in studying how the
many possible miRNA-3´UTR interactions
impact post-transcriptional regulation.

Gene of interest
RenSP
Luciferase

miRNA Mimic
RISC
complex• Understand the mechanism by which a gene is induced or repressed.
• Measure the impact of a 3´UTR on post-transcriptional gene regulation.
• Validate the targets of a miRNA or siRNA.

Light signal

LightSwitch miRNA-3´UTR product portfolio• LightSwitch miRNA Mimics and Inhibitors – Our miRNA Mimics and Inhibitors are chemically optimized synthetic double-stranded and single-stranded small RNAs, respectively.
The Mimics are used as functional equivalents to endogenous human miRNAs, while theInhibitors are used to knock down expression of endogenous human miRNAs in living cells.
• Validated 3´UTR Controls – Constructs for use as positive & negative controls.
• Empty 3´UTR reporter vector – Clone your own 3´UTR sequences.
• Complete screening service to validate 3´UTR targets for any miRNA (see other side)
For more complete information, please visit www.activemotif.com/ls-3utr.

2. Measure luciferase activity to determine
the effect on translation of miRNA binding
to the hybrid luciferase-3´UTR mRNA.

Figure 4: LightSwitch 3´UTR Reporter Assays.
A 3´UTR cloned into a LightSwitch 3´UTR Reporter
vector is co-transfected with miRNA mimics or inhibitors. The relative amounts of light produced in cellswith and without miRNA are used to measure theeffect of the miRNA-3´UTR interaction on translationof the hybrid RenSP-3´UTR mRNA transcript.
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• LightSwitch Synthetic miRNA Target Reporter Collection – These constructs contain an
optimized synthetic target consisting of sequence repeats that are fully complementary toa variety of human and viral miRNAs based on miRBase 16 annotations. These products canbe used as very sensitive screening assays for miRNA functional activity, and they also serveas strong positive knockdown controls for use in other miRNA experiments.

1. Co-transfectvector with
miRNA mimics
or inhibitors.

Hybrid mRNA

• Measure the effects of sequence variants on 3´UTR or miRNA function.

• LightSwitch 3´UTR Reporter GoClone Collection – Following systematic identificationof 3´UTR sequences in the human genome using RefSeq and other cDNA resources, over
12,000 human 3´UTRs ranging from 300 bp to 3,000 bp were cloned into the LightSwitch
3´UTR Reporter vector. Each vector is purified and sequence-verified during our extensive
quality control process, and is supplied as a transfection-ready product. Simply find andorder your clones using our online search tool (Figure 1), and then begin your experiments.

Pre-cloned intoa Reporter
VectorLightSwitch™
3´UTR
ReporterApplications for LightSwitch miRNA-3´UTR products

The LightSwitch System is ideal for performing miRNA target validation and assessing thefunctional impact of miRNA-3´UTR interactions. The 3´UTR sequence of interest is cloned
downstream of RenSP in the LightSwitch 3´UTR Reporter vector. The vector’s constitutive
promoter drives expression of a hybrid RenSP luciferase-3´UTR transcript that is the basis of
the assay (Figure 4). Total luciferase output depends on the effects that the cloned 3´UTR andmiRNAs have on the stability and/or translational efficiency of the hybrid transcript (Figure 5).

3´UTR

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Figure 5: Interaction of miR-122 with 3´UTR targets.
3´UTR targets of miR-122 were cloned into theLightSwitch 3´UTR vector and co-transfected witheither 20 nM of miR-122 mimic or a non-targeting
control miRNA in K-562 cells. The graph above shows
the four strongest responders along with two
non-responding controls.

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LightSwitch™ – comprehensive, optimized luciferase reporter system
LightSwitch™ Custom Services can help accelerate your research
In addition to the many different products that make up theLightSwitch Luciferase Assay System, we offer a wide variety ofcustom services that can accelerate your gene regulation research.

WT seed seq Mutant seed seq
AGTCTCCRIMS1 ACACTCCGNPDA2 ACACTCCA AGTCTCCAANKRD13C ACACTCC
G6PC3 ACACTCCA

ACAGACC• Leverage our expertise in cloning and reporter assays

G3BP2 ACACTCCALDOA ACACTCCA

GGTCTCC• Focus on data analysis instead of lab work
For more complete information on LightSwitch Custom Services,
please visit www.activemotif.com/ls-services.

Custom Cloning & Mutagenesis
In addition to the 30,000 human regulatory elements available aspre-cloned LightSwitch reporter vectors, we offer a custom cloning
service to quickly and economically clone any genomic elementfrom human, mouse or rat into any LightSwitch reporter vector.
We also offer a custom mutagenesis service to create sequencevariants of any element cloned into a LightSwitch vector. Custom
mutagenesis allows you to map functional motifs or characterize
the effects of sequence changes on gene expression (Figure 6).

Pathway Screening Services
Our in-house experts use our unique collection of validated reporter assays in a cell-based screening service to measure the effects ofyour compounds on a variety of biological pathways (Figure 7). We
offer 48 validated human promoter reporter vectors to measuregene expression changes associated with 11 disease-related
pathways. This service is ideal for:ACAGACCAAGGTCTCCA

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Figure 6: Mutagenesis used to identify seed sequences necessary for miR-122 function.
LightSwitch 3´UTR Reporter constructs containing the 3´UTRs of 6 genes known to bemiR-122 target sites were subjected to site-directed mutagenesis. The wild-type andmutant constructs were co-transfected in HT1080 cells with a miR-122 miRNA mimic or a
non-targeting control in triplicate and then assayed using the LightSwitch Assay Kit.

TCDD
R1881
PMA
Estradiol
DMSO
Prednisone
Heat shock
DFO
1% O2
TNF-a
IFNa
IFNg
NutlinLovastatin
Synthecol• Receive superior data validated by our control processesbe](hWj_ee\jWh][j_d]%ded#jWh][j_d]c_HD7bkY_\[hWi[WYj_l_jo

Advantages of LightSwitch Custom Services• Utilize our unique resource of promoter and 3´UTR clones

Reporterconstructs
Controls
Tox/AhrAndrogen• Secondary screening• Dose response analysis

CREB• Lead optimization• Phenotypic screening /deconvolution

Estrogen

Figure 7: Response of pathway reporter constructs to various treatments.
The heat map at right shows the inducible activity of 29 different reporter
constructs and 5 controls (rows) under 15 different conditions (columns). The
red boxes indicate promoters that are up-regulated by the condition while
the blue boxes indicate down-regulated promoters. The conditions used inthese experiments are treatments with known inducers of different biologicalpathways. For example, DFO and 1% O2 are known inducers of the hypoxia
pathway. As expected, all 3 hypoxia reporters tested are strongly inducedby both of these conditions. The heat map also shows pathways that are
activated by multiple conditions. For example, PMA and TNF-a both strongly
activate the NFkB reporters. All experiments were performed in HT1080 cells,
and the constructs are a combination of endogenous human promoters andsynthetic response elements cloned into LightSwitch reporter vectors.

Glucocorticoid
Heat shock
Hypoxia
NFkB
STAT
p53

-4

4 log2 ratio treated/untreatedwww.activemotif.com

LightSwitch™ Custom Services (cont.)
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Sequence Variant Assay Services
Naturally occurring sequence variation can have large impacts
on the function of promoters or 3´UTRs. Once you have identified a regulatory element of interest, our Custom Services
team can perform site-directed mutagenesis or custom
cloning of haplotypes, then conduct reporter assays in living
cells to quantify the functional effects of the sequence differences. Many researchers have used this service to study theeffects of naturally occurring sequence variation, while others
have created specific mutations in motifs, transcription factor
binding sites or putative miRNA targets (Figure 6).

miRNA Target Validation Services
Accurately identifying which miRNAs bind to a specific 3´UTR
can be a challenge. A given miRNA may interact with multiple
3´UTR targets, while each 3´UTR may be targeted by multiple
miRNAs. Furthermore, miRNA target prediction algorithms
suffer from low specificity and sensitivity. These factors makeit necessary to experimentally validate the targets of yourmiRNA of interest.
For this reason, we provide a complete miRNA Target
Validation Service that researchers can use to validate the3´UTR targets of specific miRNAs. Using our genome-widecollection of human 3´UTRs cloned into our optimized
LightSwitch 3´UTR Reporter vector, we can validate thetargets of miRNAs of interest using our high-throughput,
cell-based reporter assay.
Simply provide a list of candidate target genes for each ofyour miRNAs of interest, and we will perform the cell-based
reporter assays to determine whether the 3´UTRs of thesegenes are functional targets of the miRNA. If you do not havea list of candidate targets for your miRNA, we can generate
one for you (Figure 9). Our extensive set of validated positive& negative 3´UTR reporter controls enable you to analyze
your results with confidence.

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Figure 8: Measuring the functional effects of naturally occurring sequence variants.
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LightSwitch Reporter Assays were used to measure the functional differences between
two promoter haplotypes of the MIF gene (minor allele frequency of 1%). The 1 kb promoter sequences differed by a single SNP located in the predicted HIF-1a binding site
located 218 bases upstream of the transcription start of the gene. Each haplotype reporter
vector was transfected in three different cells types (HT1080, SKNSH and HCT116) andpromoter activity was measured before and after hypoxia induction (1% oxygen treatment
for 24 hours). Each vector and condition was measured in triplicate. The purple bars in the
figure show the inducible activity of the wild-type promoter sequence (major allele), while
the copper bars show a significant reduction in inducible activity in all 3 cell types tested
for the minor allele. This example illustrates the use of reporter assays for measuring thefunctional effects of naturally occurring sequence variants.

Light output with miR-122 mimic
Light output with control(%,be](hWj_ee\jWh][j_d]%ded#jWh][j_d]
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To use the service, all you need to do is to specify your regulatory elements of interest as well as the variants you want us
to create for individual SNPs or mutated motifs. We can also
analyze larger haplotypes from individual genomic samples(Figure 8). After we perform the service, you will receive botha complete raw data set and a statistical analysis of significant
differences between the variants.

(-= Effect of miR-122 on target 3´UTRFh[Z_Yj[Z) KJHjWh][ji

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Figure 9: Luciferase-based screen to identify miR-122 targets.
LightSwitch 3´UTR Reporter constructs containing the 3´UTRs of 139 genes predicted tobe miR-122 target sites were assayed in HT1080 cells for repression in response to miR-122
treatment. This barchart shows the degree of knockdown ranked highest to lowest fromleft to right. Each 3´UTR reporter vector was co-transfected with a mimic or non-targeting
control in triplicate and assayed using the LightSwitch Assay Kit. The degree of knockdown
was measured as the ratio of mimic divided by the non-targeting control.

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www.activemotif.com

With over 30,000 human regulatory elements
cloned and available as transfection-readyLightSwitch reporter vectors, Active Motif isthe leading provider of regulatory content. In
addition to pre-cloned content, our scientists
also developed and optimized every component of the LightSwitch System to ensurethat it would be easy to use and provide
optimal results.
All LightSwitch reporter vectors utilize RenSP,
an optimized marine luciferase gene that has
been engineered for maximum brightness
and short protein half-life. Starting with abase sequence of a native marine luciferase
gene, thousands of synthetic gene sequencevariants were functionally screened forincreased enzymatic activity (light output).
Furthermore, a protein destabilization

domain was added to decrease the half-lifeof the RenSP protein (see below), and putative transcription factor binding sites were
removed from the sequence, as these might
confound experimental results. The result isthat the RenSP luciferase we created is 50%
brighter than other luciferases (Figure 10).
In parallel to the protein engineering efforts,
we also created novel, proprietary companion assay reagents that provide high sensitivity over a broad dynamic range when usedwith RenSP. As an additional benefit, thereagents were developed to enable one-stepreagent addition directly to cultured cells,
eliminating the need for a separate lysis
step. Collectively, these many improvements
ensure that LightSwitch delivers superior
results with simple and robust protocols.

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Engineered RenSP luciferase and optimized LightSwitch reagents for the best results possible-'';E8gi\g(;E8gi\g);E8gi\g*

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Figure 10: The RenSP signal is brighter than hRlucP.
To determine the relative brightness of RenSP
compared to hRlucP, a humanized form of Renillaluciferase, the RenSP and hRlucP genes were cloned
into separate vectors along with the human RPL10 promoter. Three independent plasmid purifications wereconducted for each vector, and 50 ng of each plasmid
was transfected with FuGENE HD in triplicate in human
HT1080 cells using a 96-well format. After 24 hours of
incubation, the cells were assayed. The results showthat the optimized RenSP luciferase is significantly
brighter than hRlucP.

Enhanced degradation rate of RenSP improves assay responsiveness and accuracy
One limitation of other reporter proteinsis that they accumulate in the cell due to a
relatively long half-life. Slow protein turnover can decrease the measured response
to a stimulation or completely mask theability to detect repression. To minimize
this problem, the RenSP gene has been

engineered to contain a PEST protein degradation sequence from mouse Ornithine
Decarboxylase (mODC) that increases its rateof turnover. Consequently, the destabilized
RenSP luciferase protein has a half-life of
approximately one hour compared to the> 3 hour half-life of native luciferase protein.

Combined with the increased signal detailed
above, the shorter half-life of RenSP provides
a more sensitive and accurate measure of
the induction or repression of reporter geneactivity, especially when studying subtle or
fast-acting effects or when generating
high-quality dose response curves.

Single assay vs. Dual assay experimental design
Historically, some luciferase experiments used a control plasmidthat was co-transfected with the experimental construct tonormalize for variations in transfection efficiency and cell lysis
between transfection replicates. However, modern transfection
reagents such as FuGENE HD and the optimized lysis and assay
reagents found in LightSwitch Luciferase Assay Kits often reducethese types of variation to coefficients of variance (%CVs) of lessthan 10% (Table 1). Because of these improvements, normalizationby co-transfection often provides little benefit. Because dual
assay reagents cost twice as much money and can reduce assay
sensitivity, we recommend that you first try a single assay design.
However, if you are using a difficult-to-transfect cell line, theLightSwitch Dual Assay Kit is available, and has been optimized
for use with all LightSwitch reporter vectors.

Promoter

TK

ACTB RPL10 ANKRD37 EGLN1 ENO2

AVG.

%CV Single Tfx

18%

2%

13%

8%

6%

7%

9%

%CV Co-Tfx normalized 14%

22%

11%

12%

4%

7%

12%

Table 1: Variation between replicate transfections is essentially the same for single
transfections and normalized co-transfections.
Six different promoter constructs were cloned into the pLG4 vector (Promega). In the Single
Tfx experiment, they were independently transfected into Hep G2 cells in triplicate without aco-transfection control, then assayed with Steady-Glo (Promega) 24 hours after transfection.
In the Co-Tfx experiment, the same 6 constructs were co-transfected with a TK-Renilla plasmidthen assayed with Dual-Glo (Promega). The co-transfected plasmid data were normalized by
dividing the Firefly signal by the Renilla signal. The table above shows the coefficient of variation (%CV = stdev/mean) of the 3 replicates of each construct. The average %CV for the single
transfections is 9%, whereas the average %CV for the normalized co-transfections is 12%.
Similar results were seen when LightSwitch GoClones and Assays were used. (Data not shown.)

North America 877 222 9543 Europe +32 (0)2 653 0001 Japan +81 (0)3 5225 3638