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N. Kociok, B. Martiny, C. Gavranic, B. Kirchhof, A.M. Joussen; A Real Time RT-PCR-based Quantitative Comparison of Gene Expression in Central and Peripheral Human RPE Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2295.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The phenotype of AMD is characterized by retinal degeneration in the macula whereas the peripheral retina virtually remains intact. RPE cells are discussed to play a central role in this degenerating process. The different behavior of central versus peripheral RPE cells may be, at least in part, due to differences in their gene expression. Therefore, we developed a method to quantify gene specific mRNA expression in very few RPE cells isolated from different parts of the human eye in order to compare their different expression in those regions. Methods: Frozen sections of peripheral and central parts of normal human eyes were placed on 1.35 µm polyethylene membranes. Slices of 5 to 10 RPE cells were isolated 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 or random 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 gene GAPDH and the lower expressed genes PBGD (porphobilinogen deaminase), a potential additional calibration gene, PAM (protein associated with myc), a modifier of transcription, and LKHA4 (leukotriene-A4 hydrolase), a mediator of inflamation with influence on cell permeability. The output of oligo-d(T) primed or random primed cDNAs with the two used reverse transcriptases were compared. Laser captured RPE cells allow the quantification of GAPDH but not of the other three genes using oligo-d(T) primed or random primed cDNAs irrespective of the used transcriptase. Manually prepared RPE cells allow the amplification and quantification of all used genes if random primed cDNAs were used together with the reverse transcriptase included in the cell lysis kit. Conclusions: It is possible to quantify the mRNA expression of even low expressed genes in manual isolated 5 to 10 RPE cell slices using random primed reverse transcription. A comparison of gene expression in central and peripheral RPE cells is now possible and may elucidate the influence of specific gene activity that may contribute to the onset or progression of retinal degeneration diseases. Supported by DFG JO324/4-1, Köln Fortune Program/Faculty of Medicine, University of Cologne
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