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Ning Ma, Jeremy D. Rhodes, Martin C. Lott, Vincent Moulton, David C. Broadway, Julie Sanderson; A Novel Technique for Analysis of the Expression Profile of Genes in the Human Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5377.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a technique which yields high levels of good quality RNA from retinal ganglion cells for analysis of gene expression profiles in the human retina.
Human retina was obtained from the East Anglian Eye Bank within 24 hours post mortem. A 5x5mm section was dissected from the macula region of the retina and was mounted for cryosectioning. Serial 20µm sections were obtained in the plane of the retinal nuclear layers. RNA was extracted (approximately 120-360ng/section) and gene expression analysed by QRT-PCR. Markers used were recoverin (RCVRN) for photoreceptors, calbindin (CALB) for horizontal cells, choline acetyltransferase (CHAT) for amacrine cells and Thy -1, NeuN and Brn3a (THY-1, RBFOX3 and POU4F1) for retinal ganglion cells. Profiling was carried out for a minimum of 6 individual donors for each gene. Global gene expression analysis (Illumina arrays) was carried out to compare retinal ganglion cell (RGC) expression with expression in total retina (n=4). Immunohistochemistry was carried out on fixed transverse sections of the human retina.
Expression of known markers of retinal neurons was highly consistent with their position in the retina: RCVRN expression was high in the outer layers which subsequently declined to baseline levels; the second peak was for CALB and the third for CHAT; THY-1 remained highest in the latter sections derived from the inner retina. Two further retinal ganglion cell markers NeuN (RBFOX3) and Brn3a (POU4F1) showed the same profile as THY-1. The profile was confirmed using immunohistochemistry. Gene arrays, comparing the RGC layer to total retina, showed good separation between RGC and photoreceptor genes. RGC markers were amongst the set of most highly expressed genes and photoreceptor genes were amongst those with the lowest expression.
The novel technique yielded high quality RNA in sufficient quantities to carry out mRNA profiling and array analysis. The profiles of markers for retinal neurons follow the expected localisation in the retinal nuclear layers. The technique offers a powerful method for determining the expression profile of genes of interest in the human retina.
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