Dear Zhuang Xinyu ,

I have used probe based assays on occasion, but I think you can not do any meltig curve analysis with them.

The basis of a melting curve is the intercalation of the DNA-binding dye, which is (ofcause) not possible when you are using a probe.

I also think your measurement is only the noise of the detector. Correct melting curves should look different and are using a different detection technology.

Please take a look here:

https://www.researchgate.net/post/Melt-curve-analysis-using-Taqman-probe

Sönke Weinert thank you so much for you answer, actually I am using the FRET probes (one 3‘ fluorophore modified probe and one 5' quencher modified probe) to do melting curve analysis for multiplex detection of several RNA templates. this is why my melting peak looks like the red lines (downward peaks) in the figure. I also hope this NTC is only the noise of the detector.....but I also did some control experiments, when I put the polymerase and 3‘ fluorophore modified probe separately, this ntc line was smooth.so weird..... maybe I can do some normalisation work towards this NTC line with peak, but I also want to figure out the reaction mechanism...T.T

FAM is not susceptible to temperature change but to pH. The protonation state of FAM will largely affect the quantum yield of FAM.

May I ask what kind of buffer did you use for your NTC melting test? Using the temperature-independent pH buffer is critical to prevent this issue.