cross-hybridization of probes in multiplex qPCR

Use this category for questions related to various quantitative PCR methods, including real time PCR, reverse transcription (RT)/PCR, qPCR, etc. For general PCR methods, continue to use the DNA/General PCR forum

Moderators: r.rosati, Suzanne

cross-hybridization of probes in multiplex qPCR

Postby nancylowther » Apr 25 2017 4:43 am

Hi,
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.
nancylowther
newcomer
newcomer
 
Posts: 4
Joined: May 01 2016 9:59 pm

Re: cross-hybridization of probes in multiplex qPCR

Postby r.rosati » Apr 25 2017 1:10 pm

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.
r.rosati
ModSquad
ModSquad
 
Posts: 2150
Joined: Nov 04 2002 3:23 pm
Location: Brazil


Return to Real-Time qPCR/qRT-PCR Methods

Who is online

Users browsing this forum: No registered users and 5 guests

cron