What is the maximum probes could be used by Taqman PCR mutiplex? What is the advantage and disadvatage of using Taq man PCR multiplex? I have ten different genes in human DNA need to be quantified by qPCR and I have about 24 samples, so I need faster method help to do that.
Multiplexing in qPCR is difficult. There can be competition effects between amplifications, results can be unreliable and the dynamic range is much narrower. I would not gererally recommend more than duplex (actually I would only recommend singleplex). The optimization of a multiplex qPCR can be very difficult and can easily take more than a year of work. Within this time you will have quantified your samples several times using singleplex assays. Only if you have 1000s and 1000s of PCRs to run (or if you want to sell the assay), the thorough design of a multiplex qPCR can be advantagous - given the dynamic range obtained is high enough for the purpose.
If you have many target genes (100+) it may be simpler (and possibly even cheaper) to do microarrays or RNAseq.
You can do up to 4. But the more "plex" you add, The more your PCR will have chance to be unspecific. In our diagnosis laboratory we've got a triplex Real-time PCR that works just fine to detect Chlamydia trachomatis, N.Gonorrhoeae gene PorA or Opa.
Cheers
I agree with Joshen,
The most I've seen in publications were four, and usually for viruses since they have smaller genoms. However it is highly unrecommended to conduct qPCR using multiplex.
The competition among amplification can detrimentally affect your results. You may need to work separately first on optimizing a standard curve for each gene depending on its copy number in the human genom.
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