MICROPLATE READERS
DRIVE T
EFFICIENCY
Ann-Cathrin Volz explains how transfection effi ciency is measured with a microplate reader
ransfection refers to the introduction of nucleic acids into eukaryotic cells
by nonviral methods. Since the fi rst successful transfection process, using Lipofectamine, many other, mostly very diff erent methods followed. But why all that eff ort? Transfection of mammal cells is an important prerequisite for various scientifi c approaches, among them: the study of gene function, regulation, and biochemical products, silencing of genes (known to be responsible for certain disease-causing changes), mutational analysis, stable cell line generation, stem cell reprogramming, cell diff erentiation and large-scale protein production. Not every transfection method is equally compatible with every cell type and target gene. T erefore, transfection effi ciency (the ratio of cells expressing the gene of interest and all present cells) must be monitored along the transfection process to keep track of the expression yield, also depending on the applied reagent concentrations.
HOW TO MEASURE TRANSFECTION EFFICIENCY To monitor transfection effi ciency a reporter gene is often attached to the gene of interest as a proxy of its expression in the cell. Reporter genes are genes whose products can be readily assayed. T eir
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expression can either be constitutive or inducible and is commonly associated with fl uorescent and luminescent signals. T e most used reporter gene is green fl uorescent protein (GFP), emitting green light after adequate excitation. Red and yellow versions of the fl uorescent protein are also available. Next to these fl uorescent reporters, luciferases can as well be expressed with the gene of interest and then catalyse a reaction with a substrate producing yellow-green or blue light. With the blue-white screen, a bacterial lacZ gene which encodes the β-galactosidase enzyme, is introduced with the gene of interest. Upon the addition of certain galactosides, cells expressing the gene will convert the substrate to a blue product, which can be detected in a colorimetric manner. Generally, these methods are assessed manually by microscopy or single-cell
The VantaStar solution from BMG Labtech
analysis techniques, which are not only very time-consuming but also require specialised equipment such as appropriate image analysis software, making these methods very cost-intensive. Alternatively, a microplate reader capable of detecting fl uorescent, luminescent or colorimetric signals can be used for the assessment of transfection effi ciency. Typically, a second “housekeeping” signal is used to normalise to the overall cell number.
TRANSFECTION EFFICIENCY ON A MICROPLATE READER As a proof of principle for the eff ectiveness of the use of microplate readers for transfection effi ciency determination, populations with diff erent ratios of wild type (WT)-HeLa cells and GFP- and mcherry- expressing (GFP+
/mcherry+ )
HeLa cells were mixed to simulate diff erent transfection effi ciencies. 20,000 cells per
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