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2003). Calculations were made using the statistical software http://www.selleckchem.com/products/LY294002.html environment r (http://www.R-project.org). Principal component analysis (PCA), functional annotation for biological processes and identification of overrepresentation of potential transcription factor binding sites were calculated using r functions developed by the authors as previously described (Hansen et?al. 2012). In brief, the 2.5% of genes contributing the most to the first principal component were identified and overrepresentation of gene ontology (GO) terms for biological processes and of predicted transcription factor binding sites were calculated using Fisher��s exact test for proportions followed by correction for multiple testing using the Bonferroni procedure. Student��s t-test was used to compare RPE cell cultures analysed at different time points. Data were pooled for groups where no statistically significance was observed. Microarray data are reported according to the MIAME statement (Brazma http://www.selleck.cn/products/wortmannin.html et?al. 2001). Several differentially regulated genes with relation to the immune system or cell death were selected and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was performed for these candidate genes to validate the results obtained from the microarrays. cDNA was produced from 2?��g of each original RNA sample using the ��Revertaid First Strand Synthesis�� kit (Fermentas, Burlington, ON, Canada). For the qPCR reaction, a Brilliant SYBR Green QPCR Mastermix was used according to the manufacturer��s instructions (Stratagene; AH Diagnostics, Aarhus, Denmark). The reactions were run on the Stratagene Mx3000P. Reactions were performed in triplicates plus a control without reverse-transcriptase and a control without template. Table?1 shows primer sequences used. To account for differences in the amount of total RNA added to each reaction, the housekeeping gene GAPDH was used as internal amplification control. Melting curves were routinely checked to rule out the amplification of unrelated fragments during qRT-PCR. Pfaffl��s method (Pfaffl 2001) was used to calculate the relative gene expression, and mean and Standard Error of Mean (SEM) of the relative quantity of target mRNA were calculated for all groups (n?=?3). Means for all experimental groups were compared with untreated controls by one-way http://www.selleckchem.com/products/CHIR-99021.html anova and the Newman�CKeul Multiple Comparison test (Prism; GraphPad Software, San Diego, CA, USA). Values were considered statistical significantly different when p?