The level of mRNA expression in vitreous-treated RPE cells compared with untreated cells was quantified by real-time RT-PCR, by using a nucleic acid fluorescent staining system (SYBR Green I reaction system; Eurogentec, Seraing, Belgium) on a thermal cycler (iCycler; Bio-Rad Laboratories). Using the primer analysis software (Oligo, ver. 4.1; National Biosciences) gene-specific primers suitable for real-time RT-PCR were selected as shown in
Table 2 . The melting temperatures (T
m) of the primers chosen were between 58°C and 60°C wherever possible. The expected fragment length lies between 81 and 181 bp. With these primers, the mRNA expression of all genes together with
GAPDH as calibrator were analyzed simultaneously in a single experiment in triplex reactions. The analysis was repeated twice. Aliquots of the diluted cDNAs of vitreous treated or untreated human RPE cells corresponding to 10 ng initially used total RNA, were mixed with 10× reaction buffer containing Tris-HCl and KCl, 3.5 mM MgCl
2, 0.2 mM of each dNTP, 0.3 μM of each specific primer (0.15 μM for GAPDH primer), 0.5× to 1× fluorescent nucleic acid stain (Sybr Green I; Molecular Probes Europe, Leiden, Netherlands) and 1.25 U of DNA polymerase (HotGoldStar; Qiagen) in a volume of 50 μL. The following PCR cycle parameters were used: hot-start polymerase activation for 10 minutes at 95°C, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. Detection of the fluorescence product was performed during the last 10% of the cycles. To confirm amplification specificity, the PCR products from each primer pair were subjected to a melting curve analysis and subsequent agarose gel electrophoresis (data not shown). Genomic DNA contamination was excluded by control amplification reactions with nontranscribed RNA as templates. Only background fluorescence data were found. The quantification data were analyzed with the thermal cycler system software (iCycler iQ; Bio-Rad Laboratories), as described.
8 After PCR, baseline subtraction was performed by the software, the log-linear portion of the fluorescence-versus-cycle plot was extended to determine a fractional cycle number at which a threshold fluorescence was obtained (threshold cycle, C
T) for each analyzed gene and
GAPDH as the reference. Because the efficiencies of target genes and the reference gene are approximately equal (ΔC
T < 0.15), the comparative C
T method was used for quantification of the target genes relative to
GAPDH.