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. 2008 Feb 5;36(6):1792–1804. doi: 10.1093/nar/gkn005

Figure 1.

Figure 1.

Effect of DNA damage on reporter gene expression is transfected in HeLa cells. (A) Diagram of the RT-PCR analysis. The reporter gene and the PCR primers are described in the diagram. (B) Analysis of the RT-PCR products generated from RNA samples obtained at different times after UV treatment from HeLa cells transfected with the pcDNA3.1 (+)/HA actin vector. RNA samples were purified from equal numbers of cells. Equivalent amounts of RNA, quantified by optical density at 260 nm, from each sample were used in the RT-PCR reactions, which were run for 20 cycles. Equal volumes of the PCR reactions were analyzed in agarose gels. The relative density of each PCR band was determined by the Image J program. (C) Poly(A)+ RNA was isolated from cells after UV exposure, and RT-PCR (top panel) and real-time RT-PCR (bottom panel) were carried out to measure expression of GAPDH. The RT products from cells not treated with UV were used as endogenous control. Data shown are the mean ± SEM from three independent experiments. (D) Preparation of damaged pcDNA3.1 (+)/HA actin vector. Plasmids were irradiated with the UV doses indicated (lanes 4–15), treated or not treated with T4 endo V and electrophoresed on a 1% neutral agarose gel. A plasmid digested with EcoRI was used as an indicator of the linear form (6.6 kb, lane 2). Molecular size markers are also included. Positions of the relaxed (R), linear (L) and supercoiled (S) forms are indicated. (E) Analysis of the RT-PCR products obtained from RNA samples of transfected HeLa cells with plasmids treated or not treated with UV. RNA samples were purified from equivalent numbers of cells. RT-PCR reactions were performed and analyzed as in (C). (F) Analysis of truncated/polyadenylated transcripts obtained from RNA samples of transfected HeLa cells with plasmids treated (D) or not treated (ND) with UV. The poly(A) transcripts were purified as in (E) and cDNA was synthesized using an oligo(dT) primer followed by a PCR amplification with the common forward/HA primer together with the oligo(dT) primer/adapter. PCR products were analyzed by agarose gel electrophoresis.