Is anybody did digital PCR? Is there anything to pay attention to it? Is this method faster that qPCR? I will appreciate it.
Yes I also attend demo, Digital PCR is good for low expressed genes (Cq or Ct values above 30). It will take little bit more time than qPCR because first we need to generate oil droplets in this method.
Thank you, Sudhakar, do you have the protocol? would you like to share with me? How about the PCR machine and PCR kit?
Hi,
The main differences between digital PCR and qPCR are the methods of quantification. In qPCR you need to generate a standard curve, were as in dPCR the quantity of your target is worked out through Poisson distributions (eliminating the need for a standard). in dPCR the targets are partitioned into wells or droplets and these are scored as either positive or negative (for amplification), rather than measuring the change in fluorescence over each cycle.
My experience is with the droplet digital PCR (ddPCR) (Bio-Rad) rather than the chip based methods (I know Life Technologies do one), but I believe the fundamentals are the same.
ddPCR certainly provides more accurate quantification than qPCR. If you're looking for fold differences for example you can easily observe 2-fold, something that qPCR does not have the accuracy for (2-fold on TaqMan would only be a single CT difference, something that couldn't really be distinguished from experimental error).
Pros of ddPCR: no standard needed (no need to use plasmid/run the risk of contamination), assay efficiency is increased (targets are partitioned into droplets/wells on a chip so less competition), more accurate quantification, small-fold changes can be observed.
Cons of ddPCR: more expensive if you only need to test a few samples, you need a rough estimate of your target copy number before testing (you have a limited number of droplets/wells and some must be empty for the quantification to work BUT you can easily work out how much to use with a DNA titration). ddPCR is perhaps not the technique for you if you do not need a high degree of accuracy.
I've attached a paper were we used ddPCR as a starting point for you!
Hope all that helps!
EDIT: Yes, ddPCR and probably chip based do take longer to set up, but once you've got the hang of it, it's only an extra hour!
Article Germ-Line Transmitted, Chromosomally Integrated HHV-6 and Cl...
Dear Adam J Bell,
You are so kind. Thank you for sharing your experience and publication. It is really helpful. Have a good day!