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Multiplex TaqMan-like Assay PCR Efficiency - (Oct/21/2013 )

I am trying to optimize a TaqMan/Gene Expression like assay that targets three different genome areas in one assay.  Because I am technically adding only three reagents (1 reagent = primer for and rev and probe plus the obvious Universal Master Mix and genomic DNA) thus the primer and probe concentrations are fixed in the sense that the reagent has the concentrations already set and I can only adjust for amount of reagent to add.  I have been able to get the assay to work however the efficiency aka the slope is not optimal for the standard curve (-4.62 for target 1, -4.60 for target 2 and -3.83 for target 3).  I was wondering if anyone has any suggestions in getting the slope to the optimal range of -2.8 to -3.6.  The Master Mix I am currently using is the following:

Universal Master Mix 12.5 ul
Target 1 @ 10X .5ul (this includes primer and probe in one reagent)

Target 2 @ 10X .75ul (this includes primer and probe in one reagent)

Target 3 @ 10X 1ul (this includes primer and probe in one reagent)
Water                 4.75

DNA                   4 ul
total                   23.5ul


I am thinking of increasing the amount of Unviersal Master Mix to increase the amount of Taq present in the reaction.  But any other suggestions would be greatly appreciated.


Thank you in advance!


Using more Taq doesn't increase the reaction efficiency, more over using other than standard concentration of the whole Master Mix will only result in unoptimal concentration of buffer components, which is far more important.


If you want more space for the reaction, you can increase the reaction volume (and keep the Master mix in the right ratio). Reaction volumes of real-time reaction can go with no problems to 50 ul. 


Does you target mixes (probes+primers) work in defined optimum when used alone? Because if they do, the result of their failure as a multiplex results from their sequential interactions.

You actually can't just mix any primer-probe set and expect it to work perfectly every time. The initial primer-probe designs for multiplex assays have to take all the other sequences of the oligos present in account.


If it's a commercial kit, as I suppose it is, you don't even know the sequences and can't say what interacts with what (still assuming that each works fine alone), I would just ditch multiplex in this case, because it would cost you more, than to run it separately.


You can alternatively try just two of any of those together, maybe you can some of those in duplex without interference.


Apart from obvious interference in binding/alignment multiplex real-time assay have problem also with spectral crosstalk. You didn't specify what probe dyes are you using, but usualy FAM and VIC are as the first choice, and they have emission spectra quite near each other, but can be managed at least on the platforms I know (ABI, LightCycler), but the third dye is problematic, it either lies to close to the spectrum of both previous and it's more complicated to the isolate the crosstalk, or uses a more "far" dye on the spectrum, which has usually a marked decrease in fluorescence intensity level.

Multiplex real-times are in my opinion more troubles than whan can you gain by using them.




The assays do work alone very well. 


Actually this assay was previously designed and published.  However, the primers and probes were added individually instead of as one single reagent.  This process was suggested to us to limit the room for pipetting error and to make the assay easier by our sales rep.


It is not a commercialized kit but has gone through multiple validations to show that it does work, sensitive and specific.  The fluorescents being used are VIC, FAM and NED.


I fear that our choice per suggested by the sales rep (which I should have known not to rely on) was not the optimal way to go about this assay due to the lack of ability to adjust each aspect of the reagent i.e. primer vs. probe ratios.  I knew the multiplex would be an undertaking but I figured since it was shown to work previously that I would be able to accomplish this but like you suggested it may be more time consuming then need be.


I will have to consider increasing the reaction size to 50 ul.


Thank you for your was very informative.


Other ideas, sometimes problems with primer-probes mixex when added in a set instead individually, were that the interactions took place while in storage and that breaf dentaturation do 95 deg and quick cooling on ice before adding to a reaction helped. I personally encountered this problem when making a primer-probe mix instead of adding each separately from a stock, problems corrected after we denatured-cooled the mix.


Also you may give try to some real-time master mix optimised for multiplex (I've seen those.. maybe from Life Technologies.. didn't remeber), nowadays there is not a possibility to optimize mixes much.