I recently run a duplex single dye ddPCR assay to detect two genes in a single channel using the same dye (FAM). As expected, I got 3 droplet partitions, 2 single positive and one double positive line. Considering all the parameters are equal (primer/probe concentrations and annealing temp), what could be the reason for the difference in flourescence amplitute between the two genes in a single channel?
With Eva Green this can be seen with different size lengths (bp) of pcr product. Could something like this with the probe system be happening here?
Sarah Johnson I am also of the same opinion. However, i am not sure. maybe an expert in the field or rather from Bio-Rad can give us a clear answer or suggestion
If I get the question correctly, you are trying to measure 2 genes in the same channel. In general, this can be done using different concentrations of probe and primers for each target (more info: Article Fundamentals of multiplexing with digital PCR
). For example high FAM for target 1, lower concentration FAM for target 2. But there are other options that affect fluorescence amplitude then only primer/probe concentrations.The Fluorescence amplitude is determined by
- the concentration of the primers and probe. (We are looking at an endpoint reaction, once all the primers and probe are used, the fluorescence can not increase anymore). So in general, to multiplex, we advice to lower the concentration of primers and probe for the second target. (But you kept your primers and probes constant, so it is probably one of the following options.)
- the synthesis of the probe (some probes will look 'brighter' then others)
- the efficiency of your PCR (f.e. suboptimal annealing T for one of the 2 targets, ....). As long as you have 2 distinguishable populations, efficiency is not an issue.
....
I hope this helps.
Caroline Weydert Thank you so much for your comments and suggestions. I have taken them into account am grateful they have helped me learn a thing or two about the setup.
Thank you once more
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