Purchase this article with an account.
AS Hackam, T Gunatilaka, I Chowers, A Vora, M Kageyama, P Campochiaro, D Zack; Gene Expression Profiling using a Custom Murine Retinal cDNA Microarray . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2432.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify differentially expressed genes in the mouse retina, with particular interest in novel retina genes, genes involved in retinal development, and genes that contribute to retinal degeneration. Methods: A custom-made 5,376 clone set was developed representing available mouse genes known or postulated to be involved in development, neuronal degeneration and cell stress responses. Each clone was PCR amplified from a mouse eye cDNA library (J. Nathans, JHU) or from purchased IMAGE clones, purified and arrayed in duplicate onto glass slides. RNA was extracted from frozen tissue, reverse transcribed, labeled with cy3 or cy5 dyes, and then hybridized to the slides. The slides were washed and the signal was detected and quantified. Results: The array was first optimized for labeling, spotting and hybridization parameters in order to maximize signal and minimize background and non-specific hybridization. The hybridization reproducibilty was then tested by hybridizing replicate slides with RNA extracted from adult C57BL/6 mouse retina and brain. The signal expression ratios for each gene showed minimal (not significant) variation when comparing among arrays (n=8). Furthermore, RNA from rat and human retinas demonstrated strong hybridization to the mouse clone set, expanding the potential use of the arrays to other species. A subset of the array containing 550 mouse or human ESTs was used to identify novel retina genes. Hybridization of adult C57BL/6 mouse retina and brain RNA to the arrays resulted in multiple ESTs showing preferential expression in the retina, with signal ratios greater than three-fold. Of these, seven had no known homologues or recognizable motifs, making them interesting candidates for further study. Conclusion: A custom array representing the expression profile of the mouse retina has been successfully developed and has been used to identify several novel retinal genes. Together with rodent models of retinal disease, these arrays will be a valuable resource for studies of normal and abnormal retinal biology. Support: Microarray supplement from NEI and Santen Pharmaceutical.
This PDF is available to Subscribers Only