Co-Transfection Controls with Reporter Assays Luciferase Assay System

Luciferase Assay System TECHNICAL NOTE: Co-transfectionTECHNICAL NOTE: controls with reporter assays Co-transfection controls with reporter assays Luciferase Assay System

Do I need a co-transfection control? InDo most I casesneed you a doco-transfection not need a co-transfection control? control. This technical bulletin offers guidelines and experimental 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 will help you evaluate the needs of your reporter assay experiment. What does a co-transfection control do? Historically,What does an experimental a co-transfection plasmid was co-transfected control withdo? a reference or control plasmid to help normalize 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 of variation1. betweenLow and transfection variable replicates:transfection efficiency 1. 2.Low Variable and variable cell transfection lysis and variable efficiency lysate stability 2. Variable cell lysis and variable lysate stability In the days of calcium phosphate transfections and home brew assay reagents, co-transfections were essential.In the days However, of calcium modernphosphate ultra transfections high-efficiency and home transfection brew assay reagents, reagents co-transfections such as FuGENE were HD essential. and optimized However, modern lysis bufferultra high-efficiency and assay reagents transfection such reagents as the LightSwitchsuch as FuGENE Assay HD andReagent optimized often lysis reduce buffer theseand assay types reagents of variation such as the to Light- coefficientsSwitch Assay ofReagent variance often (CVs) reduce of these less typesthan of 10% variation (Table to 1). coefficients of variance (CVs) of less than 10% (Table 1).

Co-transfectionCo-transfection controls controls were were originally originally designed designed to remove to remove variability variability due to transfectiondue to transfection efficiency efficiency alone (1). aloneSuch controls(1). were Suchnot designed controls nor were are notthey designed adequate fornor comparing are they adequatebetween different for comparing conditions between or cell lines. different conditions or cell lines.

Promoter (pGL4 vector) TK ACTB RPL10 ANKRD37 EGLN1 ENO2 AVG %CV Single Tfx 18% 2% 13% 8% 6% 7% 9% (3 reps) %CV Co-Tfx normalized 14% 22% 11% 12% 4% 7% 12% (3 reps)

Table 1: Variation between replicate transfections is the same for single transfections and normalized co-transfections InTable the single 1: Variation transfection between (tfx) experiment, replicate 6 different transfections promoter-luc2P is the reportersame for constructs single weretransfections independently and transfected intonormalized HepG2 cells co-transfections without a co-transfection control in triplicate and assayed with Steady-Glo (Promega) 24 hrs after transfection. Additionally, the same 6 promoter-luc2P vectors were co-transfected with a TK-renilla plasmid and assayed withIn the Dual-Glo single transfection (Promega). (tfx) The experiment, co-transfected 6 different plasmid promoter-luc2P data were normalized reporter constructsby dividing were the independently firefly signal by transfected the renilla into signal.HepG2 The cells table without above a co-transfection shows the coefficient control in of triplicate variation and (%CV= assayed stdev/mean) with Steady-Glo of the (Promega) 3 replicates 24 hrs of eachafter transfection. construct. The averageAdditionally, %CV the for same the single 6 promoter-luc2P transfection vectorsis 9%, whereas were co-transfected the average with %CV a TK-renilla for the normalized plasmid and co-transfection assayed with Dual-Glo is 12%. (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 Aretransfection there is 9%, whereasany the down-sides average %CV for the normalized to doing co-transfection a co-transfection is 12%. control? Co-transfecting two plasmids is significantly different than transfecting a single plasmid. The effect of a co-transfectionAre there in any your experimentaldown-sides design to should doing be carefully a co-transfection considered. The most important control? considerations should be the following: Co-transfecting two plasmids is significantly different than transfecting a single plasmid. The effect of a co-transfection in your 1.experimental Time, Cost, design and Signal: should be Dual-reporter carefully considered. assay reagentsThe most important are more considerations than twice asshould expensive be the following: and take more than 2 times as long as a single transfection. A single transfection design can double the scope of a project that can1. Time, be completed Cost, and in Signal: half of Dual-reporterthe time. Furthermore, assay reagents dual-reporter are more than assay twice asreagents expensive yield and takelower more raw than signal 2 times than as reagentslong as a singlethat havetransfection. been optimizedA single transfection specifically design for can a single double reporter the scope protein of a project (Figure that 1).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).

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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 and take more than 2 times as long as a single transfection. A single transfection design can double the scope of a 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 after transfection. Additionally, the same 6 promoter-luc2P vectors were co-transfected with a TK-renilla plasmid and assayed with Dual-Glo (Promega). The data from both experiments represent raw (un-normalized) firefly luminescence. The average 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 across a 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 the experiment, 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 to measure background and non-specific responses with the highest degree of confidence.

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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 used a co-transfection control (Figure 2). If you feel like your experiment does require a co-transfection control, the LightSwitch 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 above shows the log2 ratio of the DFO treated to untreated activity for each of the 4 promoters. The fold induction for each promoter 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 and considerations 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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