Ann-Cathrin Volz explains how transfection efficiency is measured with a microplate reader
Transfection refers to the introduction of nucleic acids into eukaryotic cells by nonviral methods. Since the first successful transfection process, using Lipofectamine, many other, mostly very different methods followed. But why all that effort? Transfection of mammal cells is an important prerequisite for various scientific 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 differentiation and large-scale protein production. Not every transfection method is equally compatible with every cell type and target gene. Therefore, transfection efficiency (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 efficiency, 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. Their expression can either be constitutive or inducible and is commonly associated with fluorescent and luminescent signals. The most used reporter gene is green fluorescent protein (GFP), emitting green light after adequate excitation. Red and yellow versions of the fluorescent protein are also available. Next to these fluorescent 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 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 fluorescent, luminescent or colorimetric signals can be used for the assessment of transfection efficiency. Typically, a second “housekeeping” signal is used to normalise the overall cell number.
Transfection Efficiency On A Microplate Reader
As a proof of principle for the effectiveness of the use of microplate readers for transfection efficiency determination, populations with different ratios of wild type (WT)-HeLa cells and GFP- and mcherry- expressing (GFP+/mcherry+) HeLa cells were mixed to simulate different transfection efficiencies. 20,000 cells per well with different ratios from 0 (only WT-HeLas) to 100% (only GFP+/mcherry+-HeLas) were seeded in 96-well plates. After cellular attachment, cell nuclei of all cells were additionally stained with the fluorescent dye Hoechst33342. As displayed in figure 1, GFP, mcherry and Hoechst fluorescence signal intensity were measured on the VantaStar using a 15 x 15 matrix scan with a diameter of 6 mm. The use of the bottom optic allows to reduce background derived from auto-fluorescent medium components with one click. The included scan options thereby help to reduce data variability derived from heterogeneous samples like adherent cell cultures. The VantaStar’s LVF Monochromator provides full flexibility together with high sensitivity. For the measurement of GFP, mcherry and Hoechst33342 fluorescence intensity, the fluorophore pre-sets, available with the LVF Monochromator, were used.
As presented in Fig. 2, exemplary for GFP fluorescence detection, a linear relationship between the percentage of GFP+/mcherry+ HeLas (= transfection efficiency) and the measured signal for GFP fluorescence is observed with high accuracy (R² = 0,9997) and precision (%CV = 10.5). As visible from the consistent fluorescence intensities for Hoechst33342, the readout of stained cell nuclei reliably correlates with the total cell counts and is well suited to normalise the GFP+/mcherry+ signal.
With the Enhanced Dynamic Range (EDR) feature, the gain settings required for the best-possible fluorescent detection do not have to be determined manually in advance. With EDR each well is automatically read with the ideal gain and the measurement values are set in relation to each other without preceding manual steps. As a multi-mode reader, the VantaStar is also able to assess transfection assays based on luminescence or absorbance.
The VantaStar is an advanced detection platform to monitor transfection efficiency bypassing the need for the more time-consuming generation and evaluation of microscopic images. As a multi-mode microplate reader, it allows to precisely assess transfection assays in various detection modes.
Ann-Cathrin Volz is with BMG Labtech