Hi,
I'm starting with a gene expression study using droplet digital PCR (ddPCR). With qPCR a primer efficiency test is needed and MIQE guidelines for digital PCR (dPCR) seems to indicate that you can and it will be helpful but it is less dependent. ddPCR does absolute quantification in thousands of partitions (up to 20000). The partitions become a detection limit so you need to optimize sample concentration so that the reaction is never completely saturated (there must be negative droplets for samples). Would this then be sufficient for optimization or should I also do a primer efficiency test using qPCR for the ddPCR? I ordered new primers so I will double check annealing temperatures.
Hope you can help.
In our lab usually we test in qPCR all primers prior ddPCR in order to assure that they are at least working, and there is no contamination. However, it is better to do temperature gradient directly in ddPCR, because its optimal temperature usually 2-3C lower than that of qPCR.
Sometimes primers/probes concentrations should be titrated, espesially for duplex systems, when better clusterization is needed. Also, serial dilutions of template shows linearirt of analysis. In the case of rare mutation detection limit of detection should be determined, specifically in the case of FFPE-derived DNA.
Finally, we prefer to do rough quantification of DNA using qPCR before ddPCR experiments.
Hi Igor,
Thanks for the advice. I might do duplex, depending on how well it works with EvaGreen in the ddPCR. I'm not working with rare events and the samples were not FFPE. So check if the primers work and do complete optimization in the ddPCR? Optimize primer concentrations for clustering and template concentrations for detection limits? I don't otherwise see the need for primer efficiency test on the qPCR for ddPCR as the conditions and detection limits will differ so much between them. Please comment.
Regards,
Pieter
I suppose, in that case there is no need to do optimization in qPCR, selection of optimal conditions (primers concentrations, temperature) in ddPCR will be more effective and time-sparing. Thus, optimal primers concentrations for qPCR may be insufficient for the proper cluster separation. The latter also can be altered via annealing temperature, when EvaGreen is using.
Hope, this will help.
Yes, I agree. I also contacted technical assistance at BioRad. Optimization for ddPCR is pretty much for the same parameters as for qPCR but with a different view. ddPCR requires optimization for the right primer concentration, template consecration and annealing temperature but all with the view of remaining within detection limits, and for optimal clustering (same sample grouping) and cluster separation (differentiate genes and/or negatives). Optimization for simplex and multiplex will require you to push these parameters differently. All in all, optimizing in qPCR for ddPCR is not needed although it will or can help the optimization process in ddPCR. Advice given from Eddy van Collenburg at BioRad if qPCR data is available:
- Optimal annealing temperature in qPCR will be roughly -2C in ddPCR
- Avoid samples with Cq values
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