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M. P. Feldkaemper, R. Ashby; Microarray Analysis of Changes in Gene Expression Within the Amacrine Cell Layer Following Optical Defocus. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3834.
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© ARVO (1962-2015); The Authors (2016-present)
Ocular growth is regulated locally by signals produced in the retina that ultimately act on the growth of the sclera. Consequently, a number of studies have investigated changes in retinal gene expression during manipulation of ocular growth in an attempt to elucidate the biochemical pathways underlying eye growth. However, due to the highly heterogenous nature of the retina, important changes in gene expression can be masked. Therefore, this study has investigated changes in gene expression specifically within the amacrine cell layer, the most likely generator of growth signals, during manipulations of ocular growth.
Chicks were monocularly treated with either -7D (n=6) or +7D (n=6) lenses for 24hrs, with untreated age-matched chicks serving as a control. Total RNA from the amacrine cell layer was isolated from laser-capture microdissected (Zeiss) 10 µm thick sections. RNA quality and quantity were tested using a bioanalyzer (Agilent) and NanoDrop (Peqlabs) system. Labeled cRNA was prepared from three separate samples per condition and hybridized to Affymetrix GeneChip Chicken Genome arrays, with obtained data normalized using a GC-RMA methodology. Changes in gene expression were validated using real-time RT-PCR.
141 changes in gene expression were observed in the amacrine cell layer of minus lens treated chicks in comparison to control values of which 67 genes displayed an up-regulation and 74 genes showed a down-regulation in expression. Plus lens-treated eyes displayed 60 changes in gene expression in the amacrine cell layer following 24hrs of treatment. This was comprised of 31 up-regulated and 29 down-regulated genes. We have presently validated 2 of the gene changes observed in plus lens treated eyes (D4, zinc and double PHD fingers, family 3 (DPF3) and GDNF family receptor alpha 1 (GDNF1)) and three of the observed changes in minus lens treated eyes (DPF3, GDNF1 and hypothetical protein LOC425969) using real-time RT-PCR.
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