Quantification of the rate of recombinant emergence by real-time PCR and real-time RT-PCR. Viral RNA isolated from cell-free medium supernatant of in vitro infected NIH-3T3, HEK293, and U87-MG cells and DNA isolated from in vivo infected subcutaneous U87-MG tumours growing in Hsd:athymic Nude-Foxn1numice was used to determine the ratio of viral- and transgene-specific sequences in the packaged vector genome and in integrated proviruses, respectively. Two sets of primers and probes were used for real time PCR and real time RT-PCR, one annealing to sequences in the env gene and the second to sequences in the IRES or the eGFP gene, respectively. Viral to transgene specific sequence ratios are shown at the end of each infection cycle for cell lines infected in vitro and 5, 10, and 20 days post infection of solid tumours in vivo, respectively. Error bars represent the standard deviation of the viral to transgene ratios measured in infected solid tumours excised from 5 mice sacrificed at each time point.