Pringle, I. A., Smith, R. L., Jones, B. L., Gill, D. R. & Hyde, S. C.
Molecular Therapy, 11, S143Download Back
We are currently testing a range of non-viral gene transfer agents (GTA) in sheep and mouse models ahead of a clinical trial for Cystic Fibrosis lung disease. The selection of the GTA for the clinical trial will depend of the result of a number of assays but the accurate determination of vector derived CFTR mRNA expression is of critical importance in this assessment. Absolute levels of expression, will be determined by TaqMan RT-PCR. Assays have been designed for potential plasmid vectors containing the CMV promoter or plasmids containing the human polyubiquitin C promoter. Assays have been also developed to detect the expression levels of murine Cftr and sheep Cftr to serve as endogenous loading controls. Assays were designed to be mRNA specific by placing primers or probe sequences across intron-exon junctions. Absolute quantification of mRNA relies on the production of an RNA standard curve to quantify the levels of the unknown samples. These RNA standards or RNA mimics as they are known are required to control for the conversion of mRNA to cDNA in the reverse transcriptase reaction which precedes TaqMan PCR analysis. DNA templates for the TaqMan assays were constructed by GeneArt (Regensburg, Germany) and included the TaqMan PCR amplicon plus surrounding sequences totalling 160 bp. RNA mimics were produced using the Ambion Competitor Construction Kit, which incorporates modified ribonucleotides to the in vitro T7 RNA polymerase transcripts for increased RNase resistance. The mimics were quantified using the Molecular Probes RiboGreen assay and diluted in 50 ng/µl yeast total RNA. Dilutions were produced containing 1 to 510 starting copies/µl and 1 µl of each these dilutions was used in multiplex reverse transcriptase reactions with sequence specific primers and MultiScribe Reverse Transcriptase. TaqMan PCR was carried was then carried out using an Applied Biosystems 7700 Genetic Analyser. Standard curves were generated for the four assays. When multiplexed together, the pCI and sheep Cftr assays were linear between 100 and 1 million starting copies per reaction (R2 = 0.984 and 0.981). The UbC assay and the murine Cftr assay were multiplexed and the UbC assay was linear between 100 and 1 million starting copies (R2 = 0.987) while the murine Cftr assay was linear between 1500 and 1 million starting copies per reaction (R2 = 0.890). Control no-reverse transcriptase reactions failed to produce any amplification indicating that the mimics were free of contamination template DNA. Data will also be presented comparing the utility of absolute quantification standard curves using ssDNA oligos and unmodified RNA instead of the modified RNA mimics.