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N. Kociok, A. M. Joussen; A "Differential Expressed mRNA PCR Analysis" With No Prerequisites Reveals the Enhanced Expression of the Complement Factor H mRNA in Proliferating Cultured Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):717.
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Early passages of cultured human retinal pigment epithelial cells were used as a model system to identify differentially expressed genes without further prerequisites in proliferating retinal pigment epithelial (RPE) cells. Gene and protein expression of the identified gene were then analyzed in RPE cells.
A differential expression analysis (DEmRNA-PCR) were used to find a differentially expressed mRNA in early passages of cultured human RPE cells. The detected mRNA was identified by sequencing. Its differential expression in RPE cells was verified by semi-quantitative RT-PCR. The expression of the identified protein in vitro and its presence in surgically removed epiretinal membranes was demonstrated by western blotting and immunocytochemical analysis.
DEmRNA-PCR detected a decreased expression of a band at approximately 530 bp in human RPE cells of passage 3 (P3) compared to P0. This band was identified as part of the human complement regulatory factor H, a cofactor to complement factor I. The mRNA expression of both regulatory proteins of the complement system was confirmed in freshly prepared human RPE cells and in cultured cells from passage 0 to passage 8 by gene specific reverse transcription-PCR. Synthesis of both proteins in cultured RPE cells was verified by western blotting. The expression of both proteins in surgically removed epiretinal membranes was demonstrated by immunohistochemistry.
The identification of the differential expression of the regulatory factors H and I of the complement system in cultured RPE cells by a technique without any prerequisites demonstrates and confirms the importance of these factors in RPE cells. In addition to its known role in age-related macula degeneration our results may indicate an equal importance of the complement system in proliferative retinopathy.
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