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J.J. Diehn, K. Wong, M. Diehn, D. Sretavan; Determining the Cellular Location of Macula–Enriched Genes Identified By Cdna Microarrays . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2982.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The use of microarrays in vision research is expanding. However, information regarding expression localization of genes identified in these experiments is often lacking, thereby limiting the conclusions gleaned from the data. Because the retina is a multi–layered structure, localizing the expression of a gene with a distinctive microarray expression pattern to a particular retinal layer would be helpful in defining the biological and pathological roles of that gene. One of the goals of this project was to implement an efficient in situ hybridization (ISH) protocol for human retina tissue by utilizing the same clones used for generating cDNA microarrays. Our previous cDNA microarray studies identified a set of 486 genes that were macula–enriched (p<0.05), including a subset of genes involved in lipid metabolism. We hypothesized that the macula–enriched genes identified in the microarray experiments reflected expression in cell types that are more prevalent in the macula, such as ganglion cells. Using the protocols developed in the current project, we localized the expression of two macula–enriched genes, low–density lipoprotein receptor (LDLR) and stearoyl CoA desaturase (SCD), within human retinal tissue. Methods: Commercially available E. coli containing plasmid clones with the inserts of interest were acquired and sequence verified. These clones contained M13 primer sites that flanked T7 and T3 promoters, facilitating subsequent ISH probe synthesis. Using PCR protocols optimized for these plasmid clones, we synthesized probe template DNA, and then created Digoxigenin (DIG)–labeled sense and anti–sense probes by in vitro transcription. Following standard ISH protocols, these probes were hybridized to cryostat sections of human retinal tissue. Results: Our strategy allowed us to generate high quality ISH data to extend our microarray observations. ISH revealed LDLR and SCD expression within the ganglion cell layer. The signal was significantly more pronounced within the macula, and was minimal in the peripheral retina, thus mirroring the density pattern of ganglion cells within the macula and peripheral retina. Conclusions: Utilizing established protocols and plasmid clones used for cDNA microarray synthesis reduced the procedure time for producing viable ISH probes. The ISH experiments involving LDLR and SCD confirmed that these genes were macula–enriched, which reflected the cellular composition of the macula. Further studies may help delineate the role of lipid metabolism within the ganglion cell layer.
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