Transgene copy number comparison in recombinant mammalian cell lines: critical reflection of quantitative real-time PCR evaluation
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Nucleic acid quantification is a relevant issue for the characterization of mammalian recombinant cell lines and also for the registration of producer clones. Quantitative real-time PCR is a powerful tool to investigate nucleic acid levels but numerous different quantification strategies exist, which sometimes lead to misinterpretation of obtained qPCR data. In contrast to absolute quantification using amplicon- or plasmid standard curves, relative quantification strategies relate the gene of interest to an endogenous reference gene. The relative quantification methods also consider the amplification efficiency for the calculation of the gene copy number and thus more accurate results compared to absolute quantification methods are generated. In this study two recombinant Chinese hamster ovary cell lines were analysed for their transgene copy number using different relative quantification strategies. The individual calculation methods resulted in differences of relative gene copy numbers because efficiency calculations have strong impact on gene copy numbers. However, in context of comparing transgene copy numbers of two individual clones the influence of the calculation method is marginal. Therefore especially for the comparison of two cell lines with the identical transgene any of the relative qPCR methods was proven as powerful tool.
KeywordsCHO Cellline development Gene copy number qPCR LinReg
Part of this study was partly funded by Polymun Scientific Immunbiologische Forschung GmbH, Donaustraße 99, 3400 Klosterneuburg, Austria.
Conflict of interest
- Bustin SA (2009) A-Z of quantitative PCR. International University Line, La Jolla, CAGoogle Scholar
- Mader A, Prewein B, Zboray K, Casanova E, Kunert R (2013) Exploration of BAC versus plasmid expression vectors in recombinant CHO cells. Appl Microbiol Biotechnol 97(9):4049–4054Google Scholar
- Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386Google Scholar
- Wilhelm J, Hahn M, Pingoud A (2000) Influence of DNA target melting behavior on real-time PCR quantification. Clin Chem 46:1738–1743Google Scholar