I am going to assume that you are talking about delta-delta qPCR instead of droplet digital PCR. Let me know if this is incorrect (I am happy to talk about either).
If you are using a dsDNA dye (such as SYBR Green) you need to do a melt curve at the end of your PCR run.
Comparing the melt temperature of your PCR product to the melt temperature of your positive target control can allow you to identify if your PCR product is specific or non-specific. If the melt temperature is the same, there is a good chance that the amplification is specific. You might want to run both the sample and positive control on a gel the first time for additional confirmation.
Similarly, comparing the melt temperature to your negative target control can allow you to determine if the observed PCR amplicon is a non-specific product, such as primer-dimers.
Jakob, I never heard about a "delta-delta qPCR" (I only know the delta-delta ct method [Livak et al] for analyzing the data [ct values] obtained from rtqPCR [real-time quantitative PCR]). Therefore I assumed that ddqPCR is an unambigous abbreviation of digital-droplet quantitative PCR.
Sorry, in fact I meant the digital-droplet qPCR. Here I do not see how a gel electrophoresis or a melting curve analysis would be possible. Or is it? And if not - how can unspecific amplifications then be detected as such? Possibly a silly question, but I have no experience with ddqPCR (yet, at least).
ddPCR is what we use as an abbreviation for Droplet Digital PCR when publishing (as opposed to Chamber Digital PCR, which would be the Fluidigm and Life Technologies platforms).
I have attached a link to a paper that we did for journal club a few weeks ago on this very topic. The TL:DR is that it is assumed that the fluorescence amplitude of the droplets will be the same as your positive control for a specific amplicon and will be different for a non-specific amplicon.
How well this works in practice, I can't comment on (yet) as I have not had a chance to try it out on a QX200.
dx.doi.org/10.1021/ac403061n
Thanks you!
PS: I cannnot acces the paper. Could you send me a copy? Thanks again!
Actually, there are 2 ways you could approach this issue:
1) Run a qPCR of your assay using the ddPCR supermix. We routinely do this to check for correct amplification using a QX200 instrument.
We simply add the probes and primers to the reaction and check for late-amplification products, as they would mainly be considered non-specific. We typically analyze the data by removing the background subtraction option to ensure proper end-point fluorescence levels.
2) recover the droplets in the QX200 instrument in order to run a gel or perform other analyses.
Essentially, after the amplification (you can omit the final step at 98.C for 10 min) you can pool the reaction together - if you wish - and remove the bottom oil phase. Phenol:chlorofom works well to break the emulsion at this point and the extracted top aqueous phase contains your amplicons.
If you are indeed using a QX200, make sure you use the supermix without dUTPs, otherwise your amplicons will not stain correctly on a gel using SYBR green.
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