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Steve Huynh, Deborah Otteson; Optimizing Laser Capture Microdissection to Study Spatiotemporal Gene Expression in the Retinal Ganglion Cell Layer. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2469.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma causes retinal ganglion cell (RGC) death, resulting in irreversible vision loss. Developing therapies to replace and regenerate RGCs requires understanding gene regulation related to ganglion cell and optic nerve development. Gradients of ephrin receptors (Eph) create retinotopic maps that pattern RGC axon projections to the brain, but little is known about the regulation of these gradients. In order to test the hypothesis that nasal/temporal differences in transcription factor expressions regulate EphA5 and EphA6 mRNA gradient pattern in RGCs, this initial study focuses on optimizing laser capture microdissection (LCM) to yield high-quality RNA for transcriptome sequencing.
C57Bl/6 mice (postnatal day 2) eyes were enucleated and frozen in molds with OCT compound with or without prior sucrose cryoprotection. Eyes were cryosectioned at 7µm and mounted onto PEN-Membrane slides held at 4°C or room temperature. Sections were dehydrated prior to LCM of nasal and temporal thirds of the retinal ganglion cell layer (GCL). RNA quality was assessed using RNA Nano and Pico chips (Agilent Bioanalyzer). Nasal/temporal samples were tested in triplicate with quantitative reverse transcriptase PCR (qRT-PCR) and analyzed with Relative Expression Software Tool-MCSv2.
RNA quality was highest with flash frozen vs. sucrose cryoprotection of the eyes, with RNA Integrity Numbers (RINs) of 7.6 and 5.9 respectively (scale 0 low-10 high). Mounting sections on 4°C slides yielded better RNA quality than on room temperature slides, with RINs of 8.8 and 7.8 respectively. Minimal RNA degradation was detected within 90 mins following dehydration, with RINs declining from 8.8 to 8.5. qRT-PCR showed about a 3.5-fold enrichment of Pou4f2 mRNA in LCM samples vs. whole retina. There was about a 2-fold enrichment of EphA5 mRNA in temporal GCL vs. nasal GCL.
LCM provides a powerful technique to extract high-quality RNA with sufficient yields for downstream, high-content sequencing. Histological sections and RT-qPCR support an enrichment of RGC mRNA. Nasal/temporal differences in EphA5 were detectable. Additional samples will be collected under optimized conditions and analyzed prior to whole transcriptome analysis of the nasal and temporal GCL.
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