If you have you used the QX100 or QX200 system, or perhaps another digital PCR system, what do you think? What would you improve? What are some frequently asked questions?
Hi Alexander. I am having problems with varying levels of background fluorescence. Essentially, for some of my splenic RNA, the amplitude of the negative population will be around 5,000 and for others it is around 15,000. My positive control droplets are in the range of 10,000, so it makes results interpretation impossible. Do you have any suggestions?
Anna - check out our paper on handling bulk samples with high background signal (DOI: 10.1139/cjfr-2019-0047) wherein we address a similar issue.
This is a comment regarding the analysis software not the actual ddPCR technology/system itself... but it would be really helpful to be able to change the colors assigned to the different droplet populations after you gate them in the Quantasoft program. The default colors that the software assigns are really difficult to see and inconsistent between wells (as in, if I gate something as positive in one channel for a particular well, then move on and gate that same population for a different well it chooses another color, even though they represent the same thing - very annoying) - and, to the best of my knowledge, there is currently no way to change them. Everyone in my lab (as well as collaborators outside of our lab) who I have talked to about this issue agrees and considers it to be the most major flaw in the Quantasoft program and a persistent annoyance when trying to share images of ddPCR plots for presentations, grants, etc.
Hi. I recently tried a duplex single dye (FAM) based assay to detect two genes in a single channel. As expected four partitions were seen, double negative, two single positives and double positive droplets. It has been said before that varying the concentrations can increase or decrease the droplet amplitudes of a particular gene. But for my assay, under same concentrations of primers and probe, there is a total difference between the fluorescence amplitude and location of the two genes. What may be the reason for this separation?
Alexander King
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?
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