Rapid titer determination using quantitative real-time PCR

N. Sanburn, K. Cornetta

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

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 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 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.

Original languageEnglish (US)
Pages (from-to)1340-1345
Number of pages6
JournalGene Therapy
Volume6
Issue number7
DOIs
StatePublished - Jul 1 1999

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Keywords

  • Quantitative PCR
  • Retroviral vectors
  • Vector production
  • Vector titer

ASJC Scopus subject areas

  • Genetics

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