Rapid titer determination using quantitative real-time PCR

Quantitative real-time PCR was utilized to evaluate retroviral vector titer. RNA was prepared from vector supernatant and run in a one-step Rf-PCR reaction combining reverse transcription (RT) and amplification in one tube. Sample analysis was performed in the ABI Prism 7700 Sequence Detector. PCR was quantitative over a range of 10(1) to 6 x 10(5) vector particles per reaction (2 x 10(2) to 1 x 10(7) vector-particles per millilites of supernatant) and closely correlated with biologic titers performed on the test material. The 96-well capacity of the machine and 2 h of running time permit titer determinations within 8 h, facilitating the processing of large sample numbers while greatly decreasing technician time. real-time PCR imporves titer quantification and the identification of high-titer producer cells. This methodology will help investigators meet the challenges of developing vectors which lack selectable markers.