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 RT-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 101 to 6 x 105 vector particles per reaction (2 x 102 to 1 x 107 vector particles per millilites of supernatant) and closely corre- lated 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 improves 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.
Sanburn
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Indiana University Vector Production Facility, Department of Medicine, Indiana University, Indianapolis, IN 46202-5121, USA.
Cornetta
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D005822:Genetic Vectors; D012190:Retroviridae; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014773:Virology / Q000379:methods