cross-hybridization of probes in multiplex qPCR

Go to the profile of nancylowther
Apr 25, 2017

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.


Go to the profile of Roberto Rosati
Roberto Rosati 9 months ago

Having more details could help...
For each of the two probes, do you see a marked difference in Cq between the two targets, or are they totally unspecific i.e. the Cq is very similar for target A and target B?
When you say that in silico the two probes are specific, which software or what parameters did you use to check them?
It would really help if you made explicit which papers are using the probes, so other users might take a look and judge by themselves to advise on the matter; but it's ok if you don't want to.