Improvement of RNA stability by combination of the optimized structural features. (A) Immature dendritic cells were transfected with different eGFP variants featuring combinations of the improved structural characteristics. Cells were harvested 48 hours after transfection, and the eGFP transcript level was assessed by real time RT-PCR. Cells electroporated with buffer or with RNase-digested RNA were used as controls and reference. Transcript levels were shown relative to expression levels obtained for eGFP-2β-globinUTR-A(120). (B) Fluorescence microscopy of mature dendritic cells 24 hours after transfection with standard eGFP-A(67)ACUAG and optimized eGFP-2β-globinUTR-A(120) IVT RNA. To allow comparison, images were obtained using equal acquisition parameters. Images were taken with an Olympus-IX71 inverted microscope (Hamburg, Germany) with a 20×/0.4 NA objective lens. TILLvisION software (TILL Photonics, Gräfeling, Germany) was used for image acquisition. (C) Immature and mature dendritic cells were transfected with different IVT RNA constructs encoding for the short-lived d2eGFP variant. Mean fluorescence intensities of viable cells were determined at different time points after transfection in 3 independent experiments. Data for both cell types are shown as the mean ± SEM of 3 experiments.