Digital PCR workflow and products

You may refer to our QIAcuity Application Guide for detailed guidelines and step-by-step tutorials on how to set up digital PCR experiments. We would also like you to refer to the digital MIQE guidelines and their update (Huggett JF et al, 2013 and 2020) for additional considerations when setting up and reporting results from digital PCR experiments.

The QIAcuity reads emitted fluorescence from the bottom of the plate, which is covered with a foil. For best results, keep the foil clean and avoid damages such as scratches. Also, keep the barcode on the side of the plate clean and intact. Ensure that you wear gloves when working with a plate and do not apply force to it. For safe handling of the plate, please place the plate into a nanoplate tray.

Your dPCR system software will likely provide you with first-level and second-level analyses. First-level analysis could include a concentration diagram, a view of the partitions in wells of interest or a heatmap to compare the target channel to a reference channel. You can use histograms and scatterplots to change threshold settings. In second-level analysis, you can analyze PCR data according to application aims (mutation detection, gene expression analysis, etc.).

Designing primers for digital PCR is similar to designing primers for qPCR. You should pay attention to target matching, base composition, amplicon length, melting temperature, secondary structures, self- and inter-complementarity (self-annealing) and cross reactivity. One difference is that primers for dPCR are often used at higher concentrations than qPCR to better separate specific signals from background noise.

For the vast majority of QIAcuity dPCR applications, template DNA is uniformly distributed throughout the QIAcuity Nanoplate reaction chambers. In QIAcuity reactions using PCR products or highly fragmented DNA (FFPE DNA, circulating cell-free DNA), complementary DNA (cDNA), gBlocks, etc., as a template, a uniform distribution of PCR signal is observed. However, DNA molecules >20 kb or plasmids are unevenly partitioned, which leads to over quantification of template concentration. By adding restriction enzymes directly to the QIAcuity reaction mixes, large templates can be fragmented to smaller sizes, which results in even template distribution and accurate quantification. This is particularly important for copy number variation (CNV) analyses, where multiple copies of a gene might be linked in tandem. When adding restriction enzymes to reaction mixes, users must be sure that the enzymes do not cut within the amplicon sequence. It is recommended to include restriction enzyme in the master mix and incubate for 10 min at room temperature after template addition.

The QIAcuity dPCR instrument can detect dyes such as FAM, EvaGreen, VIC, HEX, TAMRA, ROX and Cy5. 

All DNA samples used in reaction mixes should show similar quality and quantity, which can easily be assessed using UV spectrophotometry. DNA samples with an average length of ≥20 kb (e.g., genomic DNA purified via spin column with silica-membrane) should be fragmented by restriction digestion before partitioning. Enzymatic fragmentation of larger DNA ensures an even distribution of template throughout the QIAcuity Nanoplate, which in turn leads to an accurate and precise quantification.