cross-hybridization of probes in multiplex qPCR
I am trying to develop a duplex real-time PCR and have used primers and probes that have been successfully used according to 3 different research papers. I have target A and target B that share a common forward and reverse primer pair. Probe X is specific to target A and Probe Y is specific to target B according to in silico analysis. I did an experiment to see if there was any cross-hybridization of probes to non-target DNA and I found that Probe X binds to both target A and target B and Probe Y binds to both target A and target B. I am doing the expt. in FAST mode on the ABI 7500, and the other studies did the assay in Standard mode. Any ideas?
Thanks in advance.