Dear community,
I am planning to perform a multiplex PCR with seven different probes, however, there are limited quencher options. I want to understand how, having same quencher for 2-3 different fluorophore probes impact the PCR quantification? Also, till what wavelength range of fluorophores (a company offers 439 to 800nm) function well in the reaction?
Anyone has any suggestion on how to choose 7 different fluorophores other than they should be 25-30nm apart in absorption wavelength?
It is not necessary to use a quencher for each reporter fluorophore. A single quencher is usually sufficient to mask the signal of multiple fluorophores over a relatively wide range of wavelengths. At the following link you will find a tool to evaluate possible combinations of fluorophores and quenchers, it is even possible to evaluate the best combinations per thermal cycler.
https://www.biosearchtech.com/qpcr-multiplex-spectral-overlay-tool
It is important to note that the ability to multiplex 7 targets is also determined by the instrument. It will be necessary to know the excitation-emission pairs of the detection channels available in the instrument.
It may be necessary to calibrate the instrument for certain fluorophores.
Dear Jyoti,
if you really want to detect 7 different targets in one assay I suggest that you set up 2 different mastermixes with 4 and 3 targets, respectively. Then you can choose the best dyes or channels for your instrument and make sure that you have a minimum of cross-over.
Regards
Dirk