Figure 4

Half-life analysis of the intron locus transcripts. Mouse fibroblasts were infected with MCMV (MOI = 1.0). At 30 hours post infection, cells were treated with 4 ug/ml of Actinomycin D. Total RNA was harvested over the indicated time course. Intron, mRNA, and 18 srRNA transcript levels were quantified by qRT-PCR. Intron and spliced mRNA transcripts were normalized to 18 srRNA. The relative quantitative values at time zero hours were adjusted to 100% and transcript remaining was compared relative to time zero. The fitted curve was modeled by one phase decay using a non-linear regression analysis on four biological replicates for each time point. The half-life (t1/2) shown for each transcript is the best-fit value. Bars represent the mean and error bars represent the standard error of the mean (SEM). (A) Schematic representation of primer probe sets used for qRT-PCR analysis. SD = splice donor sequence, SA = splice acceptor sequence. Half-life decay curves for the (B) 7.2 kb intron using primer probe set a/a’, (C) the second exon of the mRNA (b/b’), (D) the spliced mRNA using primer probe set c/c’, and (E) GAPDH.