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Christopher M Aderman, Ye Sun, Zhuo Shao, Jean-Sebastien Joyal, Lois Smith; Novel technique for sectioning a mouse retinal flat mount. Invest. Ophthalmol. Vis. Sci. 2014;55(13):407.
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There are currently no reliable, established methods that allow for isolation of retinal layers in order to enrich for tissue or cell-specific genes for microarray analysis. Existing techniques, such as laser capture microdissection, yield very small quantities of genetic material that must undergo amplification, a step which invariably induces error and can make microarray studies unreliable. We have developed a new method to section a flat mounted retina so that sufficient quantities of tissue specific genes and proteins can be studied in normal and diseased states.
We sacrificed neonatal mice at postnatal day 12 and harvested the eyes. We then dissected the retinas in saline and made four radial cuts extending from the optic nerve to allow the retina to lay flat. We performed the dissections on Parafilm-covered microscope slides, then flipped over the retinas onto pre-frozen and trimmed blocks of OCT. The exposed portions of the retinas were embedded in OCT and prepared for sectioning. Optimal sectioning size was approximately 20 microns, which allowed for a sufficient tissue quantities while still allowing for resolution of the retinal layers. High quality RNA was converted to cDNA for real time PCR analysis.
The left panel is a schematic showing how the flat mounted retina is sectioned at a thickness of 20 microns, which provides multiple sections per retinal layer. The right panel compares Rhodopsin (a photoreceptor marker) mRNA expression in each of the 12 sections normalized to the housekeeping gene, cyclophillin A. There is clear enrichment or rhodopsin expression in layers 1 and 2 (54 million and 78 million copies, respectively per 1 million copies cyclophillin A), which indicates these layers correspond to photoreceptor layers and represent a 3.8 to 12 fold increase compared with the other retinal sections.
This novel technique for flat mounting and sectioning fresh mouse retinas serves as a powerful tool for RNA (or protein) analysis. The primary advantage of this new method is that we are able to obtain large amounts of high quality RNA and protein from specific retinal layers, making large scale, cell-type specific, gene expression analyses of normal and diseased retinas feasible.
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