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R.K. Shuler, M.J. Phillips, A. Fernandes, J.H. Boatright, A.Y. Chow, J.M. Nickerson, M.T. Pardue; Initial Growth Factor Expression Profile in the RCS Rat Retina . Invest. Ophthalmol. Vis. Sci. 2004;45(13):705.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Subretinal implantation of prosthetic, silicon chips has been demonstrated to have neuroprotective properties in the RCS rat retina (Pardue et al., 2003). We plan to test whether this is due to growth factor regulation in the RCS rat retina. The purpose of the current study was to develop a baseline expression profile of eight growth factors in the RCS rat retina that will allow future comparisons to expression levels after subretinal implantation. Methods: RCS rats were sacrificed at eleven weeks of age and total RNA was extracted from retina and choroid. Real–time, reverse–transcriptase PCR (RT–RT–PCR) was conducted using primers for the following genes: aFGF, BDNF, bFGF, bNGF, CNTF, GDNF, HGF, NTF3, and rhodopsin. Reactions were run on four independent eyes. Primers were designed to span introns. Amplifications were normalized against reactions using 18S and GAPDH primers. Expected product sequences were queried against the NCBI non–redundant ESTs database. Melt curve analyses were run on reaction products. RT–RT–PCR products were electrophoresed across 1% agrose gels to determine product length. Results: BLASTN query of the NCBI non–redundant ESTs database demonstrated no significant matches other than the expected gene products. RT–RT–PCR demonstrated curves that crossed threshold at the following mean cycle numbers: aFGF 32.8+/–1.2, BDNF 32.2+/–0.8, bFGF 24.0+/–0.5, CNTF 27.8+/–0.1, HGF 28.8+/–0.5, NTF3 30.6+/–0.5, and rhodopsin 21.5+/–0.6 cycles (mean +/– SD). GDNF and bNGF signals were not detected. Melt curve analyses demonstrated similar products for independent reactions. Gel electrophoresis demonstrated reaction products of the expected length for aFGF, BDNF, bFGF, CNTF, HGF, NTF3, and rhodopsin. Conclusions: The primers designed appeared to quantitate the expression levels of the desired neurotrophic genes. The growth factors bFGF, CNTF, HGF, NTF3, BDNF, and aFGF (but not GDNF and bNGF) appear to be expressed in the RCS rat retina. Compared to rhodopsin expression, the neurotrophic factors appear to be expressed at significant levels.
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