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C.Y. Kim, M.H. Kuehn, A.F. Clark, Y.H. Kwon; Laser Capture Microdissection and Microarray Analysis of the Human Retinal Ganglion Cell Layer . Invest. Ophthalmol. Vis. Sci. 2006;47(13):410.
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© ARVO (1962-2015); The Authors (2016-present)
Cell death in the retinal ganglion cell layer (GCL) is a prominent feature in glaucoma. The purpose of this study was to identify and catalogue the genes whose expression is restricted to the GCL in the human retina.
Laser capture microdissection (LCM) technology was used to isolate tissue from the perimacular region of three normal human donor retinae. The tissue was preserved within four hours postmortem. Material was isolated from the (1) retinal ganglion cell layer and (2) the inner and outer nuclear layers of the same retina. RNA was extracted, amplified, and the gene expression profiles of both fractions were determined using Affymetrix Hu133Plus 2.0 GeneChips. Data were evaluated by two–class paired statistical analysis to identify those genes whose expression is considerably substantially more prevalent in the GCL when compared to the outer retinal layers.
The results show that mRNA levels of previously described ganglion cell markers were highly enriched in the isolated GCL fraction. Examples include neurofilament genes (NEFH, NEF3 or NEFL), the Brn3b transcription factor (POU4F2) and the beta 3 subunit of the nicotinic cholinergic receptor (CHRNB3). In contrast, transcripts for genes associated with phototransduction (PDE6A, RDH12 or OPN1SW), photoreceptor development (NRL) or interphotoreceptor matrix constituents (IMPG1) were nearly absent from the GCL fraction. Approximately 30 genes were identified whose expression in the human retina appears to be limited to the GCL.
We have successfully used LCM technology to generate gene expression profiles of highly enriched GCL as well as outer retinal fractions of the normal human retina. The results will serve as a resource in the development of ganglion cell specific markers or transfection vectors and the identification of candidate genes for hereditary forms of glaucoma.
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