two probes: FAM and VIC
when I run a assay in singleplex qPCR reaction, the Ct(s) value is good,about 23. when I perform the assay added two probes in one reaction, the Ct(s) separately is higher than singleplex, about 30.
Does anyone have any advice on it? my confusion is whether the Ct(s) must be the same between singleplex and duplex.
In this situation, when we test samples, the results are liable or not.
My recommendation would be first to compare the characteristics of the primers and probes using a software program with an algorithm that will 1) predict melting temperature and %GC for each of the primers and probes and make sure that they match the cycling parameters that you are using, and 2) check for self-dimers and cross-dimers among the primer and probe sequences. The Primer Probe Test Tool in Primer Express is a good example. Also, be sure that you have adequate nucleotides, primers, and polymerase (e.g., to excess).
thank you for your suggestions
redesigning the primers and probes may be the best answer to solve my problem .
thank you .
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