Impact of regulatory components located 3′ of the coding region on transcript stability and protein yield in dendritic cells and cell lines. (A) Influence of poly(A) tail length on transcript stability in iDCs and mDCs. Cells were electroporated with equal amounts of d2eGFP-encoding IVT RNA species, which differed in the lengths of their poly(A) tails. Cells were harvested after 6 hours, 24 hours, 48 hours, and 96 hours, and eGFP transcript levels were quantified by real-time RT-PCR. Cells electroporated without RNA served as controls. For each time point, the transcript levels (+SEM) are shown relative to expression levels obtained for d2eGFP-2β-globinUTR-A(120) in iDCs. (B) Flow cytometric analysis of protein levels in the same cells used in the experiment described in panel A, which were transfected with the short-lived d2eGFP variant as reporter molecule. (C) Cells were transfected with eGFP-encoding IVT RNA variants differing in the lengths of their poly(A) tails. RNase-digested RNA and untailed RNA served as controls. Cells were harvested after 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 144 hours, and 192 hours, and eGFP fluorescence of viable cells was measured by flow cytometry. (D) Influence of the 3′ untranslated region (UTR) on translational efficiency. Immature and mature dendritic cells were electroporated with eGFP RNA variants differing in their 3′ UTR. Cells were harvested after 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 144 hours, 168 hours, or 218 hours, and the eGFP fluorescence of viable cells was measured by flow cytometry.