Correlation of mRNA delivery timing and protein expression in lipid-based transfection

A. Reiser; D. Woschée; N. Mehrotra; R. Krzyszton; H. H. Strey; J. O. Rädler

Combined phase contrast and fluorescence images that were taken 24 h after the cells' transfection (see Figure 3A ) show clear differences regarding the intensity of the fluorescent cells as well as the number of eGFP positive cells. The contrast settings of the combined phase contrast and eGFP fluorescence images are kept constant enabling a direct comparison of intensities. Furthermore, we used these endpoint images to determine the fraction of eGFP positive cells as measure for the transfection efficiency. The transfection efficiency decreases from 92% ± 3% for cells transfected without serum to 31% ± 6% for cells transfected with 10% FBS (data shown for cells treated with 0%, 2%, and 10% FBS in Figure 3A ). Similar behavior of decreasing transfection efficiency of lipid-based vectors in the presence of serum was observed previously (23, 24) . The corresponding fluorescence time courses in Figure 3B show the single-cell expression time courses together with their respective mean time course. We observed a large difference in the translation kinetics for cells transfected without serum compared to cells transfected with serum, which is in agreement with the changing transfection efficiency. The influence of serum is also reflected in the decreasing number of analyzed cells per condition, which is expected due to the decreasing transfection efficiency assuming that we have approximately the same total number of cells per condition.