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Specifically, quantitative real-time RT-PCR on the iCycler iQ was performed in duplicate or triplicate on 1 l of template cDNA per 20 l reaction. Mix A reactions consisted of PCR buffer (16.6 mM (NH4)2SO4, 67 mM Tris, pH 8.8, 6.7 mM MgCl2, 10 mM β-mercaptoethanol; Loging et al. 2000), 1 mM dNTPs (Invitrogen), 0.5 U of Platinum Taq DNA polymerase (Invitrogen), 10 nM fluorescein calibration dye (Bio-Rad), 1 l of a 1:1,500 dilution of 10,000x SYBR Green I stock, 500 nM of each GSP, and 1 l of cDNA. iQ supermix reactions consisted of iQ supermix (Bio-Rad) at a final concentration of 1x, 10 nM fluorescein calibration dye, 1 l of a 1:1,500 dilution of 10,000x SYBR Green I stock, 500 nM of each GSP, and 1 l of cDNA. To control for pipetting losses, 19 l of each 20 l reaction was amplified in a 96- well thin-wall PCR plate (Bio-Rad) using the following PCR parameters: 95C for 2 min followed by 50 cycles of 95C for 15 sec, 60C for 15 sec, and 72C for 15 sec. Melt-curve analysis was performed immediately following amplification by increasing the temperature in 0.4C increments starting at 65C for 85 cycles of 10 sec each. The presence of a single PCR product was verified both by the presence of a single melting temperature peak representing a specific product (vs. a nonspecific primer-dimer peak) using iCycler iQ analysis software and by detection of a single band of the expected size on a 4.5% Super AcrylAgarose gel.
Real-time RT-PCR was performed in duplicate or triplicate reactions.
Each GSP pair was used with each reaction mix on each of the two different
cDNA templates (derived from macula or periphery). Real-time RT-PCR reactions
for detection of the endogenous control gene, A
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