A beginner’s guide to dPCR – Find tips and resources for bringing it to your lab and getting started
Nanoplate dPCR basics
The QIAcuity Nanoplate dPCR workflow involves three basic steps: pipetting and loading, running the experiment and analyzing results. In principle, the concept is similar to qPCR. The critical difference is in the precision and sensitivity gain while maintaining the cost of quantification.
Step 1: Prepare and load
As a first step, it is important to get your samples ready. We recommend measuring DNA amount and purity. Depending on your application, the sample can also be tested in different dilutions. It is advantageous to use pure samples without high amounts of PCR inhibitors. For more information regarding sample preparation, refer to the QIAcuity Applications Guide.
Next, create an experiment in the QIAcuity Software Suite by defining the dPCR parameters – priming, cycling and imaging profiles, reaction mixes, samples & controls and plate layout. Once the plate has been defined, it is ready for a run. Prepare the reaction mix in a pre-plate by combining the QIAcuity PCR Master Mix with the sample, primers, RNase-free water and optionally restriction enzyme. The final reaction volume depends on the QIAcuity Nanoplate used. The prepared reaction mix can then be pipetted into the nanoplate. To prevent evaporation and contamination the nanoplate should be sealed properly. A roller is used to fix the foil on the plate macrostructure. The proper fixing of the plate seal by manual rolling is important for a good filling result. It is critical to hold the plate at the side edges or by the tray and transport it to the QIAcuity smoothly without shaking or turning to ensure that the reaction mix is at the bottom of the input well. When the instrument and suite are connected, all modules are ready for use and plates can be loaded and started.
Step 2: Run setup and amplify
Once the plate is loaded, the instrument will highlight the slot in blue and scan the barcode. The preferred experiment previously set up in the Control Software can be linked to the plate, the priming, cycling and imaging profile defined and run initiated.
First, the plate is processed in the priming/rolling module. At this step, the reaction mix of each well is partitioned into a thousand little individual reactions. Then PCR is performed in a thermocycler. The suitable template material within one or the other partition will lead to a positive fluorescence signal which is detected during imaging. The images are sent to the QIAcuity Software Suite for image processing.
One can view the current plate status either on the instrument screen or in the Software Suite.
Step 3: Analyze results
To analyze, select one of the following: Absolute Quantification, Mutation Detection, Genome Editing, Copy Number variation or Gene expression. For Absolute Quantification Analysis, just select the preferred wells and either target or detection channel. The list view shows the concentration, confidence interval as well as valid positive and negative partitions. A heatmap view shows the target channel alongside the reference channel. Use either histogram, 1D scatterplot or 2D scatterplot to change threshold settings. Click “recalculate” to get results based on the changed threshold.
