Focus: Reporter Assays
Normalizing Genetic Reporter Assays: Approaches and Considerations for Increasing Consistency and Statistical Significance
Genetic reporter assays are used to study DNA sequences and cellular processes that control gene expression. Reporter activity is used as an indicator of the ability of a test sequence cloned upstream of a reporter protein-coding sequence to regulate gene expression under the experimental conditions. Reporter activity is compared between different vector constructs (e.g., deletion analysis of promoter sequences; see reference 1) or treatment conditions (e.g., screening for G-protein-coupled receptor pathway modulators; see reference 2). Because variables such as cell number and transfection efficiency can have an unwanted effect on the magnitude of reporter expression, reporter data should be normalized. This article discusses approaches for normalization and highlights some key considerations for successful data analysis.
By Trista Schagat, Ph.D., Aileen Paguio, M.S., and Kevin Kopish, B.S.,
Promega Corporation
Published in April 2008
What is Normalization?
Normalization is a process by which data are corrected for factors other than those being directly tested in the experiment. To normalize reporter data, the reporter activity in a particular sample is divided by a second value specific to the same sample. The primary purpose of normalization is to remove sample-to-sample variability caused by factors other than those being tested in the experiment. These factors can include variabilities in cell plating and transfection efficiency, pipetting inconsistencies, and toxicity. Data from each sample is normalized prior to making comparisons between test groups. Data normalization reduces variability and allows data comparisons to be made with greater confidence.
| Normalization Method | |||
|---|---|---|---|
| None | Protein | Vector | |
| Calculation | None | Reporter Activity | Reporter Activity |
| Total Protein | Control Reporter Activity | ||
| Assays | Reporter | Reporter and Protein | Two Reporters |
| Ideal Use(s) | Estimations; Some Stable Transfections |
Stably Transfected Cells | Transiently Transfected Cells |
| Removes Variability Due To |
Not Applicable | Cell Number | Transfection Efficiency |
What are the Advantages of Vector Normalization?
Vector normalization controls for differences in transfection efficiency between samples. By factoring in transfection efficiency, vector normalization reduces data variability and can give differences between test groups greater statistical significance. Figure 1 shows the effect of the normalization method on analysis of three different promoter constructs. When comparing samples with large differences in reporter activity, such as constructs A and C, the vector normalization method gives a greater fold change compared to the other analysis methods. The coefficient of variation, which gives a measure of data variability, was significantly lower for vector-normalized results.
Figure 1. Effect of normalization method on data significance and variability. pGL4.10[luc2] (firefly luciferase) reporter vector containing one of three different promoters (constructs A, B or C) was cotransfected into HEK 293 cells with the pGL4.74[hRluc/TK] (Renilla luciferase) control vector. Firefly and Renilla luciferase activities and protein concentrations were determined using the Dual-Luciferase® Reporter Assay System (Cat.# E1960) and the Coomassie® Plus Bradford Reagent (Pierce), respectively. Firefly luciferase data were either not normalized (None), or normalized to total protein (Protein) or Renilla luciferase activity (Vector). Panel A. Firefly luciferase activity for each normalization method was calculated as fold change compared to Construct A. Data represent the average ± standard deviation of triplicate samples. Panel B. The coefficient of variation (standard deviation/average) was calculated for the normalized construct data, and the average ± standard deviation of all the constructs is shown for each normalization method.
How Do I Optimize Transfections to Include the Internal Vector Control?
The optimal amount of control vector to use in cotransfections is the minimum amount that gives significant reporter activity above background (background is measured in samples transfected with only the test vector). This must be determined empirically. Using optimized transfection conditions for a single reporter (for general guidance, see reference 3), cotransfect varying amounts of control vector. Typically a ratio of 10:1 test vector:control vector is used; however, as little as 100:1 ratio of test:control vector may be sufficient. The amount of vector needed depends on the control promoter and the cell line.
Figure 2 gives an example of the effect of internal vector control concentration on expression of both test (firefly) and control (Renilla) reporters. It is important to ensure that the presence of the internal control does not affect test reporter activity. As shown by the 1:1 and 1:2 ratios in Figure 2, too much internal control vector decreased the expression of the test reporter. For this reason, minimal concentrations of the control vector are used.
Figure 2. Effect of varying concentrations of internal control (Renilla luciferase) vector on test reporter (firefly luciferase) activity. CHO cells were transfected with a constant amount of pGL3 Control Vector expressing firefly luciferase (50ng) and varying amounts (0–100ng) of the internal control vector, phRL-SV40 (expressing Renilla luciferase). Total DNA concentration was held constant using carrier DNA. Firefly (Panel A) and Renilla (Panel B) luciferase activities were assayed using the Dual-Luciferase® Reporter Assay System. Data represent the average ± standard deviation of triplicate samples.
| Vector | Multiple Cloning Region | Reporter Gene | Reporter Gene Promoter |
|---|---|---|---|
| pGL4.10[luc2] | Yes | luc2 (firefly) | No |
| pGL4.13[luc2/SV40] | No | luc2 (firefly) | SV40 |
| pGL4.70[hRluc] | Yes | hRluc (Renilla) | No |
| pGL4.73[hRluc/SV40] | No | hRluc (Renilla) | SV40 |
| pGL4.74[hRluc/TK] | No | hRluc (Renilla) | HSV-TK |
| pGL4.75[hRluc/CMV] | No | hRluc (Renilla) | CMV |
| For a complete listing of all pGL4 Vectors, go to: www.promega.com/pGL4/ | |||
Summary
Data normalization should be considered for all genetic reporter assays. Because of the variability inherent in transient transfections, normalization to an internal vector control should always be used. The ideal control vector will give low to medium expression and consistent results under all test conditions. Normalization is performed for each sample prior to making comparisons between test groups. Using vector normalization, variability is reduced, consistency is increased, and greater significance between samples can be achieved. Additional information can be found in the Cell Notes 17 article of the same title.
Reference
- Figueiredo, M.S. and Brownlee, G.G. (1995) cis-acting elements and transcription factors involved in the promoter activity of the human factor VIII gene. J. Biol. Chem. 270, 11828–38.
- Paguio, A. et al. (2006) Using luciferase reporter assays to ccreen for GPCR modulators Cell Notes 16, 22–25.
- Chapter 12: Bioluminescence Reporters, Protocols and Applications Guide (2006) Promega Corporation www.promega.com/paguide/chap12.htm
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