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Abstract
Abstract
Comparison of Gene Expression in Central and Peripheral Human RPE Cells by Real Time RT-PCR
Kociok N., Kirchhof B., Joussen A. M.
University of Cologne, Ophthalmology, Department of Vitreoretinal Surgery, Angiogenesis Laboratory, Cologne
Purpose: In AMD retinal degeneration occurs in the macula whereas the peripheral retina virtually remains intact. In this degenerating process RPE cells are discussed to play a central role. At least in part, the different behavior of central versus peripheral RPE cells may be due to differences in their gene expression. In order to compare the different expression in those regions, we developed a method to quantify gene specific mRNA expression in very few RPE cells isolated from different parts of the human eye.
Method: Frozen sections of peripheral and central parts of normal human donor eyes were placed on 1.35 mm polyethylene membranes. Slices of 5 to 10 RPE cells were cut off into reaction tubes by laser microdissection or manually under the microscope. After cell lysis and DNAse treatment reverse transcription reactions were performed without an additional RNA isolation step using two different RNA reverse transcriptases. For priming oligo-d(T) primers, random primers, or gene-specific primers were used. The GAPDH-calibrated gene expression of the analyzed genes were quantified using a SybrGreen I -based Real Time RT-PCR analysis.
Results: The technique was tested on four genes known to be expressed in RPE cells: the highly expressed house-keeping gene GAPDH (glyceraldehyd-3-phosphat deh