Quantifying non-viral gene transfer by real-time PCR: a cautionary tale.

Coles R, Stoneham C, McLachlan G, Vrettou C, Gill DR, Hyde SC, Sumner-Jones SG

Human Gene Therapy, Vol 20, 11, Abstract P265

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The European Society of Gene and Cell Therapy Conference, Hannover, 2009

Q-PCR is a powerful tool for measuring gene transfer in cells and tissues, which we have used to measure gene delivery (DNA) and expression (mRNA) from plasmid vectors in lung tissues. Here, we report a biochemical anomaly that can lead to the detection of false positives after non-viral vector mediated delivery of plasmid DNA in animal models.

A quantitative RT-PCR TaqMan assay (pCI-A) was designed across an intron boundary in a standard plasmid that had been aerosolized to sheep lungs and results expressed as % VE (% Vector/Endogenous CFTR mRNA). Initial results were encouraging, with vector-specific expression levels of 1510 % VE at day 1. Omission of reverse transcriptase (RT) from the assay resulted in negligible signal, thus the pCI-A assay was judged insensitive to DNA contamination.

Unexpectedly, we then observed that RNase treatment of the total RNA samples did not result in a significant reduction in vector mRNA levels, whereas DNase treatment reduced RT-dependent TaqMan signal to

New primer sets were designed, which showed that the DNA- and RT-dependent signal was observed only when a specific reverse gene primer was used. In conclusion, our experience suggests that caution should be exercised when developing new TaqMan assays, particularly where DNA may contaminate samples expressing low levels of RNA.