Does high ROX levels invalidates a qPCR assay ?

Go to the profile of egrau
Mar 29, 2017

Hi all,

We are having problems with ROX levels (higher than SYBR Green when visualized the Multicomponent plot) when using some qPCR chemistries. We know there are different formulations not interexchangeables for different qPCR devices. My question is:
How these anormal high levels of ROX do affect the Ct obtained ? Do they simply reduce the sensibility of the assay ? Do they fully invalidate the results ?

Thanks in advance,


Go to the profile of Roberto Rosati
Roberto Rosati 6 months ago

On the bleed-through front, ROX and SYBR emit at very different wavelengths, and you shouldn't see much bleed-through from the ROX channel to the SYBR channel.
But if the ROX signal is too much and goes beyond the linear range of the instrument, then data normalization will be faulty... in this case, yes, a high ROX level can invalidate the assay. Specifics will depend on the machine you have.

Go to the profile of egrau
egrau 6 months ago

Thanks for your time rosati and all those who are always willing to help !