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M.P. Fautsch, K.G. Howell, A.A. Leonovich, S. Raghavakaimal, D.H. Johnson; Global Gene Expression in Primary Trabecular Cell Monolayer Culture Is Altered Following Incubation in Human Aqueous Humor . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1843.
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To determine if incubation of primary trabecular monolayer cells in human aqueous humor (the normal nutritional source in vivo) has an effect on global gene expression when compared to cells incubated in the standard culture media containing fetal bovine serum.
Primary monolayer trabecular cell lines were grown to confluency in 10% FBS (n=4 cell lines). Cells were washed and placed in either 10% FBS or 50% human aqueous humor (obtained from human donor eyes within 12 hours of death). Cells were maintained at 37° C in 5% CO2 for 5 days. After incubation, cells were harvested and total RNA was isolated using Qiagen RNeasy total RNA isolation kit. Total RNA was used to probe an Affymetrix GeneChip Human Genome U133 Plus 2.0 DNA microarray. Following normalization, differentially expressed sequences were determined based on a call of present in the initial analysis and a ≥ two–fold change in all pair wise comparisons. Several of the differentially expressed genes were verified by real–time PCR to determine accuracy of the analysis. Differentially expressed genes were classified by function and Ingenuity Pathway Analysis program was used to visualize and explore relevant gene regulatory networks.
Over 400 genes were differentially expressed when human trabecular cells incubated in human aqueous humor were compared to those incubated in fetal bovine serum. Genes involved in various cellular functions were affected including cell signaling, cellular proliferation, protein synthesis, and the cell cycle. Real time PCR of 10 differentially expressed genes verified the accuracy of the microarray analysis. Analysis of differentially expressed genes in aqueous humor incubated cells suggests a role for cell cycle inhibitors p27Kip1 and p57Kip2 in trabecular cell quiescence.
Significant differences in gene expression are present in primary trabecular monolayer cell cultures incubated in human aqueous humor when compared with fetal bovine serum. Culture conditions that mimic aqueous humor are essential to make molecular and biochemical studies physiologically relevant.
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