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Kieron Torres, Mariam S. Assadian, Verity F. Oliver, Ray A. Enke, Shannath L. Merbs; Isolation Of Cell Layer-specific DNA From Human Eyes With Glaucoma Or AMD. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5985.
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Little is known about the relationship between DNA methylation patterns, retinal gene expression, and retinal disease. In the US alone, almost 4 million individuals currently have either primary open angle glaucoma (POAG) or age-related macular degeneration (AMD). With both diseases, gene expression changes in the retina have been observed. One modulator of gene expression is DNA methylation. We hypothesize that alterations in DNA methylation, accompany and may actually precede the gene expression changes seen with the onset of POAG and AMD and are working towards a genome-wide map of the DNA methylation changes in the human retina associated with these two diseases. High quality, cell layer-specific DNA from diseased and control eyes is required for our genomic analysis.
Adult human eyes, enucleated within 6hrs after autopsy and transported on ice within 24hrs after enucleation, are obtained from NDRI. Eyes are from individuals with AMD, POAG, or without history of either disease. The optic nerve is cut sharply with a razor blade, placed in 4% paraformaldehyde, and embedded in plastic. The cornea is removed. Four radial cuts are made in the quadrants between rectus muscles and are extended to the equator of the globe. A high-resolution photograph is taken of the macula and optic nerve. The eye is put through a 6.25-25% sucrose gradient on ice over 2hrs. A 6mm trephine is used to isolate the macula and optic nerve head in separate calottes. The calottes are flash frozen in a 2:1 mixture of 25% sucrose:OCT and cryosectioned.
Cryosections through the optic nerve head, as well as plastic sections though the optic nerve, are used to confirm axonal loss in the POAG samples and exclude axonal loss in control eyes. Gross examination of the macular photographs and cryosections of the macular calottes are used to confirm or exclude the diagnosis of AMD. LCM of cryosections on PEN membrane slides is used to collect ganglion cells from POAG eyes and photoreceptors and RPE cells from AMD eyes, as well as all 3 cell layers from control eyes. DNA and RNA are isolated with AllPrep (Qiagen). Pyrosequencing of the rhodopsin gene, and QPCR of ganglion cell-, photoreceptor cell- and RPE-specific genes, have demonstrated our ability to isolate quality, cell layer-specific DNA and RNA.
Using cryosectioning and microdissection of a 6mm macular calotte, we are able to obtain quality, cell layer-specific DNA and RNA from the ganglion cell, photoreceptor and RPE cell layers. The DNA will be used for a pangenomic epigenetic analysis to identify DNA methylation changes associated with AMD and POAG.
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