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. 2013 Sep 17;2(9):e122. doi: 10.1038/mtna.2013.49

Figure 4.

Figure 4

Development of integrated provirus-specific real-time PCR system. (a) To reduce overestimation of vector copy number (VCN) per cell due to residual plasmids, we designed self-inactivating long terminal repeat (SIN-LTR)-specific probe/primers. (b) In tenfold dose escalation of GFP-encoding plasmid DNA (upper) or provirus-integrated DNA (VCN = 1, lower), the SIN-LTR–specific probe/primers detect only provirus signals (concentration 0.1–100 ng, lower), and not detect plasmid signals (0.01–100 pg, upper). The GFP-specific probe/primers detect both the integrated provirus and the plasmid signals. (c) Addition of >100 pg plasmids (comparable to 3.8 × 103 copy number per cell) into provirus DNA (10 ng) resulted in increase of SIN-LTR signals and reduction of ribosomal RNA (rRNA) signals. Ct, threshold cycle; F, forward primer; GFP, green fluorescent protein; No signals, signals are less than threshold for detection at 40 cycles; P, probe; R, reverse primer.