Accurate estimation of transduction efficiency necessitates a multiplex real-time PCR

Transduction efficiency can be easily monitored during preclinical trials b y inclusion of marker genes. However, the use of such marker genes should b e avoided in the final clinical gene therapy application since their produc ts are often immunogenic, making it difficult to monitor transduction, espe cially if the vector is applied in vivo. In these cases PCR-based methods l ike the real-time PGR might provide a powerful tool to estimate biodistribu tion. To investigate the accuracy of this method, we have developed and tes ted a real-time PCR assay for the quantification of the enhanced green fluo rescent protein (EGFP) gene and compared the results with transduction effi ciencies estimated by FACS analysis. Although our real-time PCR assay itsel f was characterized by a high precision over a wide dynamic range of quanti fication, significant differences in the transduction efficiency compared w ith FACS data were initially observed Accurate determination could only be achieved using an optimized multiplex real-time PCR assay, which allows the simultaneous calculation of cell number and EGFP copy number in the same t ube. In view of future needs for methods allowing precise and accurate anal ysis of biodistribution in gene therapy trials, our data highlight the nece ssity critically to check both parameters in the implemented assay.