FIGURE 4.

The presence of the 3′-UTR-CARE is associated with differential priming of reverse transcription by oligo(dT) and random hexamers. a, total cytoplasmic RNA extracts from transient transfections of HeLa cells with specified pcDNA 3.1-based vectors were analyzed by quantitative PCR using luciferase primers following reverse transcription with either oligo(dT) or random hexamers. Poly(A) purification was omitted. The 3′-UTR CARE and the CUCARE+, but not the CURE alone, affected the measurable amount of input luciferase RNA as a function of priming with increased levels seen with random hexamers. Data are shown as a percentage of input RNA in control transfection. b, summary of effect of CARE on input luciferase mRNA measured by quantitative PCR following priming shown as random hexamers relative to oligo(dT) (n = 8).c, cytoplasmic RNA was analyzed by quantitative PCR following priming of reverse transcription with oligo(dT), RHs, or a luciferase-specific primer. Data are presented as a ratio of the effect of reverse transcription primer or luciferase-specific primer on apparent input RNA relative to that seen with oligo(dT). The 3′-UTR CARE increased the ratio of input luciferase mRNA with both luciferase-specific priming and random hexamers by 4-fold relative to that seen with oligo(dT). In contrast, little or no effect was seen on luciferase mRNA from cells transfected with control and 3′-UTR-CURE-containing plasmids.