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Jay Ching-Chieh Wang, Jiangyuan Gao, Sijia Cao, Idris Samad, Dean Zhang, Orson L. Moritz, Jing Z. Cui, Joanne A. Matsubara; Comparison of Genomic DNA Isolated From Iris And Retina of Postmortem Eye Tissues. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1574.
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© ARVO (1962-2015); The Authors (2016-present)
Recent genomic developments have propelled our understanding of mechanisms underlying complex eye diseases such as AMD to the genetic level. Genotyping postmortem eye tissues for known SNPs associated with AMD may prove valuable, especially if combined with other methods such as immunohistochemistry and western blot analysis on the same donor eye. Since AMD tissue samples are rare, using nonretinal tissues to genotype would preserve the bulk of the retinal tissues for these other forms of analysis. Since no previous evidence is available, this study assesses the quality and quantity of DNA obtained from iris and retina of postmortem eyes for genotyping studies.
Thirty-four pairs of postmortem eyes consented for research and obtained from the Eye Bank of BC were used in this study. Iris and retinal tissues of approximately 3mm x 3mm x 3mm were extracted from one globe of each pair and genomic DNA was isolated using a standard phenol-chloroform-isoamyl (PCI) and alcohol method. The quantity, purity, and quality of the DNA examples were examined using a Nanodrop spectrophotometer and gel electrophoresis. A 506 bp fragment of the Complement Factor H (CFH) gene containing the Y402H SNP previously shown to be associated with AMD was amplified via PCR. Amplification was quantified by agarose gel electrophoresis using a GelDoc imaging unit and ImageJ software. The results of each tests were used to compare the quality and quantity of amplified DNA obtained from the iris and the retina.
Genomic DNA was successfully isolated from retina and iris of all 34 eye samples. On average, the final concentration of DNA obtained from retina was greater than that obtained from iris (936ng/uL vs. 78ng/uL, p<0.01). Average 260/280 ratio of 1.78 and 1.46 were reported for retinal and iris DNA respectively (p<0.01), suggesting that retinal DNA had less protein contamination. ImageJ analysis following gel electrophoresis showed an approximately 60% greater yield of PCR product for retina vs. iris DNA (p<0.01). Genotyping was possible through direct sequencing of iris and retinal PCR products, but retinal products yielded superior results.
Both iris and retinal genomic DNA isolated using the PCI method from postmortem eye tissues are suitable for PCR amplification and sequencing, allowing for CFH genotyping. If both tissues are available, retinal tissue is the preferred choice in terms of quantity and quality of DNA.
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