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Real-Time Immuno-PCR on the iCycler iQ System, Rev A

g/L with phosphate buffer containing BSA and immunoglobulins to reduce nonspecific adsorption (CanAg PSA EIA instructions).

To each well, 25 l of the diluted biotinylated anti-PSA66 was added and the samples were incubated for 1 hr at room temperature. After washing six times with wash buffer 1, the wells were incubated for 30 min at room temperature with 25 l streptavidin solution (0.3 g/ml streptavidin in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 2.5 mM MgCl2). Thereafter, the samples were washed six times with wash buffer 1 and incubated with 25 l of biotinylated DNA marker (15 pM). The wells were then washed six times with wash buffer 1, followed by four times with wash buffer 2 (0.154 M NaCl, 5 mM Tris, pH 7.75), and then left to stand in wash buffer 2 for 1 hr before removing the buffer. Finally, the PCR mix was added to the wells and real-time PCR was performed in the iCycler iQ system (Figure 2). Approximately 1520 cycles were needed to reach threshold, set at 10,000 relative fluorescence units (RFU), which was substantially above the background but still in the exponential growth phase of the PCR. The CT vs. log (concentration) plot was linear with a large correlation coefficient (r = 0.986), showing that the assay correctly and accurately reflects the amount of PSA in the studied range. However, PCR products that appear in negative control samples limit sensitivity. Analysis revealed two sources of these products. One is formation of primer-dimers, which form independently of the presence of template. There are several ways to suppress their formation through assay optimization techniques. The second source of error is nonspecific adsorption of the detection antibody or DNA. This i
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