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T.J. McFarland, Y. Zhang, L. Atchaneeyasakul, J.T. Stout, B. Appukuttan; Realtime qRT–PCR to Compare Polyadenylation With the Soluble Neuropilin–1 or the SV40 Polyadenylation Signals . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5265.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The use of large genes (like soluble receptors) for the treatment of ocular neovascular disease is limited by the carrying capacity of most vectors. We have previously shown that the soluble Neuropilin–1 (sNRP–1) gene employs an extremely short polyadenylation (pA) signal that is efficient in producing gene transcripts capable of producing protein (hrGFP). However the stability and copy number of mRNA carrying this novel pA signal is unkown. We aim to quantitatively compare the levels of transcripts containing the sNRP–1 pA signal with those carrying the SV40 pA signal. Methods: 293T cells were transiently transfected with either pUC18–CMV–hrGFP–SV40pA or pUC18–CMV–hrGFP–sNRP–1pA via a CaPO4 transfection protocol. RNA was isolated using the RNAaqueous–4PCR kit (Ambion) and quantitated by the Ribogreen method (Molecular Probes). 200ng of RNA was reverse transcribed (RT) using the iScript cDNA synthesis kit (Bio–Rad) following manufacturers protocol. RT mix was added to the HS SYBR green quantitative PCR master mix (MJR) using 500uM hrGFP primer concentrations. The optimized PCR reaction was performed on a Chromo4 realtime thermocylcer (MJR). A standard curve was made from serially diluted pUC18–CMV–hrGFP–SV40 plasmid ranging from 0.032fg to 320pg. Data was analyzed using OpticonMonitor software (MJR). Results: Threshold cycle (Ct) values for the SV40pA and sNRP1–pA were 29.02 and 29.84, respectively. These values were converted to pg based on the standard curve (R=0.998). No significant difference (P=0.57) was observed. This data suggests that the overall starting copy number for equally loaded cDNA were relatively equal in abundance from the two samples. Conclusions: This data suggests little to no difference between the levels mRNA generated by the sNRP–1 polyadenylation signal and the SV40 polyadenylation signal, despite a remarkable signal size difference. The sNRP–1 pA is a viable candidate for use in gene therapy vectors. The 200–300 bp reduction in the pA signal could potentially increase the payload of gene therapy vectors.
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