I am conducting a multiplex Real time PCR using FAM (Rotavirus A), VIC (Rotavirus C) and Cy5 (18s internal control) dyes in Applied Biosystem 7500. After PCR when I try to analyze turning FAM, VIC and Cy5 detectors on, the Ct values for all three targets are different when I analyze turning only 2 or 1 detector on. For example first time if I get the Ct for Rota A: 24 (all detectors on), second time Ct for Rota A: 22 (same sample, FAM and CY5 on). The threshold level I select same (0.2) in both analysis. The Passive reference dye ROX was on in all analysis. I will appreciate any comments/suggestion regarding this issue.

Thanks,

Niaz