mRNA cellular persistence: quantitative RT-PCR of Non-viral (A and B) and Truncated-retroviral (C and D) mRNA. 293FT cells were transfected with constructs. One day after transfection, transcription was blocked by actinomycin D, and luciferase-mRNA levels were measured at 24, 48 and 72 h. Luciferase mRNA was normalized to that of actin. (A and C) Comparison of luciferase mRNA level in transfected cells, for each condition the amount was normalized to that of b-actin in cells transfected with pcDNA-Luc-psi or pCMV-5′LTR-psi-Luc and pcDNA-Luc-psi-UTR or pCMV-5′LTR-psi-Luc-UTR. The bars indicate relative amounts compared to control condition (in A pcDNA-Luc-psi and in C pCMV-5′LTR-psi-Luc). Number of experiments, N = 2. (B and D) Evolution of the specific amount of the different RNAs. RNA extracted from cells immediately after the addition of actinomycin D (time-point 0) was used to define the initial level of mRNA and arbitrarily set to 100%, at 24, 48 and 72 hours the amount of the different RNAs are compared to this initial point. Number of experiments, N = 2.