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The amplification signals were detected in real time, which enabled accurate quantification of the amounts of the initial RNA template. Two oligonucleotides with differently labelled fluorophores were hybridized to the amplified fragment during [http://www.selleckchem.com Dinaciclib] the annealing phase. When the two probes came in close proximity, fluorescence resonance energy transfer developed between the two fluorophores. The emitted fluorescence was then measured by the LightCycler instrument. Hybridization probes were displaced during the extension step. Depending on the initial concentration of target genes, the signal intensity increased in different cycles, and these cycles were used as the crossing point. The standard curve was made with three different probes of untreated RPE cells. For MMP-1 mRNA signals were detected at a mean of 24 cycles (��3 cycles), for MMP-3 mRNA signals were detected at a mean of 21 cycles (��4 cycles). To normalize for differences in the amount of total RNA added to each reaction, 18S rRNA was simultaneously processed in the same sample as an internal control. The level of MMP-1 and MMP-3 mRNA was determined as the relative ratio (expressed in decimal format), which was calculated by dividing the level of MMP-1 and MMP-3 mRNA by the level of the 18S rRNA housekeeping gene in the same samples. All experiments were performed at least in triplicate and repeated three times. All data were analysed with SPSS 13.0 for Windows (SPSS, Chicago, IL, USA). For  all statistical tests,  P?