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D.V. Ivanov, G. Dvoriantchikova, L. Nathanson, V.I. Shestopalov; Microarray Profiling of Gene Expression in Purified Adult RGCs . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5500.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGCs) transfer visual information to the brain and are susceptible to selective degeneration in various neuropathies, like glaucoma. Such a selective vulnerability suggests that survival of these highly specialized neurons is dependent upon a distinct gene expression profile that becomes altered by a neuropathy–associated stress, thus facilitating RGC death. In this work we used comparative microarray analysis to determine the genes with expression profiles unique to RGCs.
To label the probes for microarray, the RNA was isolated directly from the adult RGCs that were purified by immunopanning without sub–cultivation in vitro. This allowed us to avoid alterations of the original gene expression profile by cell culture conditions. To identify genes that were enriched in adult RGCs relative to other retina cell types, we compared global transcriptional profiles of purified primary RGCs to that of the whole retina and selected genes with the expression differences at a 2–fold cutoff level.
From a total of 8,069 transcripts detected in RGCs by microarrays we identified 326 genes enriched in these RGC neurons and 330 genes enriched in the whole retina. The microarray results were selectively verified by qRT–PCR, in situ hybridization and immunohistochemistry. Our RGC–derived set of genes had 874 (76% of those present on the arrays) genes common with the previously published EST–derived dataset. Analysis of the main Gene Ontology (GO) categories revealed that genes vital for neurogenesis and survival were enriched in RGCs. Some of these genes predominantly expressed in RGCs like Nrg1, Rgn, 14–3–3 family (Ywhah, Ywhaz, Ywhab), Nrn1, Gap43, Vsnl1 and Rgs4 may serve as novel markers for these neurons.
This approach allows gene expression profiling of a small number of purified adult RGCs. Among the genes enriched in RGCs we identified potential novel RGC markers and genes essential for survival and homeostasis in these cells. We detected elevated expression of the genes controlling pro–survival pathways in RGCs relative to other retina cells.
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