Transfection efficiency can be calculated by counting the number of cells transfected over the total cells in a particular sample. The number of cells can be counted by two methods. The most frequently used method is to use easily tractable reporters. These reporters can be green fluorescence protein (GFP), luciferase, beta-galactosidase, secreted alkaline phosphatase (SEAP). Most recent method to calculate transfection efficiency is to label nucleic acids, enabling tracking intracellular delivery of them. Some other approaches such as Western blotting, immunostaining, and functional assays can also be used to calculate transfection efficiency. Since transfection efficiency depends on multiple experimental parameters, the determination of transfection efficiency is the key to determine the influence of various factors on transfection.
Key Areas Covered
1. What are the Methods Used to Calculate Transfection Efficiency
– Reporter Systems
2. How To Calculate Transfection Efficiency
– Cell Counting
Key Terms: Flow Cytometry, GFP, Nucleic Acid Labeling, Reporter System, Transfection Efficiency
What are the Methods Used to Calculate Transfection Efficiency
Different types of methods are used in to calculate transfection efficiency.
1. Reporter Systems
– Green fluorescence protein (GFP) – GFP is naturally found in jellyfish. It is used for both qualitative and quantitative analysis of transfected cells by simultaneous transfection of the GFP gene with the gene of interest. Qualitative analysis can be done under a fluorescence microscope. Quantitative analysis can be done by flow cytometry. In addition to GFP, red fluorescence protein (RFP), and yellow fluorescence protein (YFP) can also be used as reporters. E. coli colonies expressing fluorescent proteins is shown in figure 1.
Figure 1: Fluorescent Proteins
- Luciferase – Luciferase is naturally found in fireflies, generating light. Luciferase assays are highly sensitive and can be used for the quantitative analysis of transfected DNA.
- Beta-galactosidase – Beta-galactosidase is found in E. coli. It is used in the qualitative analysis done with X-gal and the quantitative analysis done by colorimetric, fluorescent or chemiluminescent methods.
- Secreted alkaline phosphatase (SEAP) – SEAP is a heat stable reporter, which is secreted from mammalian cells when expressed.
2. Nucleic Acid Labeling – Fluorescently-labeled plasmids can be used in the transfection. Then they can be counted under a fluorescence microscope.
3. Western blotting, immunostaining, and other functional assays
How to Calculate Transfection Efficiency
The number of cells that exhibit the reporter and the number of cells that do not exhibit the reporter can be counted under a microscope or by flow cytometry. The percentage of cells that exhibit the reporter gives the transfection efficiency.
Figure 2: Transfection Efficiency
Conclusion
Transfection efficiency can be calculated by determining the number of cells that exhibit transfected DNA over the total number of cells in the sample. The number of cells can be calculated under the microscope or by flow cytometry using a reporter system.
Reference:
1. “Measure Transfection Efficiency.” Mirus Bio LLC, Available here.
Image Courtesy:
1. “E. coli expressing fluorescent proteins” By Erin Rod – Own work, (CC BY-SA 4.0) via Commons Wikimedia
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