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Tips from the Bench: Transfection Tip

Measure Transfection Efficiency

Transfection efficiency can be affected by multiple experimental parameters. To determine how these factors influence transfection, a sensitive and robust detection assay is required. Researchers frequently employ easily tractable reporter assays for determining transfection efficiency and their downstream applications. An alternative and more direct method is to label nucleic acids to track their intracellular delivery (see Track Nucleic Acid Delivery Intracellularly). Other approaches include western blotting, immunostaining, and functional assays, etc.

Reporter Systems

Commonly used reporter assays for measuring transfection efficiency include:

Green Fluorescent Protein (GFP)—An inherently fluorescent protein often found in marine organisms, such as jellyfish. Since the discovery of GFP, several other fluorescent reporters have been introduced; e.g. yellow fluorescent protein (YFP).

  1. Qualitative analysis - Cells expressing GFP can be visualized directly by fluorescence microscopy. Visual assessment is quick but can be biased depending on the field of view.
  2. Quantitative measurement - Flow cytometry enables the measurement of GFP expression in a large population of cells with high sensitivity and represents the most accurate and objective method for determining transfection efficiency.

Luciferase—An enzyme found in many organisms, e.g. firefly, that catalyzes the production of light in a reaction involving luciferin and ATP. The luciferase assay allows for a quantitative readout of photon emission. Luciferase assays are highly sensitive with a broad dynamic range, making them ideal for determining relative transfection performance between samples. Most luciferase assays require cell lysis.

β-galactosidase (β-gal)—An E. coli enzyme encoded by the LacZ gene.

  1. Qualitative analysis - Cells can be treated with the precipitating colorimetric substrate X-gal, which when cleaved by β-gal will stain the cells blue allowing visual assesment.
  2. Quantitative measurement - The availablity of various substrates allows colorimetric, fluorescent, or chemiluminescent detection of β-gal. These formats provide high sensitivity detection but require cell lysis.

Secreted Alkaline Phosphatase (SEAP)—A modified heat stable reporter that, when expressed, is secreted from mammalian cells. Cells transfected with SEAP expression constructs do not need to be lysed to assay for activity. Since lysis is not required, culture media can be sampled at multiple times throughout an experiment to generate a time-course. SEAP assays can be colorimetric or fluorescent in nature.

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