I'm planning to try multiplex qPCR. I found the following description in the past questions:
"Beware. Multiplex quantification is very tricky with dozens of pitfalls that will lead to false results. You have to be an expert in PCR design to make it work well."
(https://www.researchgate.net/post/Can_primers_developed_for_SYBR_Green_PCR_be_used_with_TaqMan)
Do you agree that multiplex qPCR is very tricky, if yes, how so? What should I expect (what those companies don't mention when marketing their products)?
Hi Yulia,
I agree that Multiplexing is more challenging than single plex applications.
The challenges here are that you have more primers and probes in one reaction and the targets are differing in abundance. Therefore it is important to be sure, that the chemistry is capable of quantitative amplification of these targets even with strong abundance differences. In addition you need to check, that the systems are not interfering with each other.
In general in my hands Multiplexing does work well as long as the assays are also performing good as singleplex and the chemistry is capable for Multiplexing
Best Sven
I agree with Sven Reister for using multiplex qPCR. It depends on how many primers and probes together you would like to use and how much differences persist among them in the expression abundance as well as the chemistry is capable to quantify them or not.
the main problem is when you use different primer with different reaction conditions, so is beter using primers that similar in reaction condition, other else multiplex not recommended .
KR.
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