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E. Chaum, H. Yang, X. Yang, J.C. Lang; Tissue Culture Wash Conditions Significantly Alter Gene Expression in Cultured Human Retinal Pigment Epithelial Cells – A Real Time RT–PCR Study . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3096.
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Purpose:Studies of oxidative stress (OS) in the RPE have previously demonstrated OS–induced changes in the expression of genes including transcription factors and genes regulating the redox status of the cell. Many of these studies include a washout of the OS followed by temporal analyses of gene expression. The aim of our study was to examine the effects of tissue culture manipulation on gene expression in human RPE cells in vitro. Methods: Confluent ARPE–19 cells were cultured for three days in defined NR–1 medium to stabilize gene expression. RNA was isolated from the cells at 0, 1, 4, and 24 hours following different wash conditions. These included: i) no touch control, ii) manual pipette aspiration, iii) vacuum aspiration followed by washing, and iv) a modified manual pipette aspiration. RNA was isolated at each time point and gene expression was quantified using real–time RT–PCR. Results:We examined genes FosB, JunB, EGR–1, and the antioxidant enzyme heme oxygenase–1 (HO–1). Standard vacuum aspiration followed by washing induced very large quantitative changes in the expression of FosB, JunB, and EGR–1 within one hour. The peak level of induction at one hour changed the Ct value by as much as 6–10 cycles, depending on the gene. This effect was significantly reduced by more gentle washing and media replacement methods but these still induced changes in expression of between 2–4 Ct cycles. All genes returned to basal levels of expression after 24 hours. Only by eliminating all manipulations were all method–induced changes in genes expression eliminated. Having established the requisite experimental methods, we determined and will report on the effects of oxidative stress on these and other genes in the RPE. Conclusions: Routine tissue culture methods can induce significant changes in gene expression in human RPE cell cultures. This may be due to mechanical sheer stress, transient cell drying, changes in osmolarity or other causes. The potential confounding effects of these culture methods used in studies of OS must be carefully controlled in order to accurately determine the OS–specific effects being studied.
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