Preparing your sample for digital PCR
Digital PCR (dPCR) is a polymerase chain reaction (PCR) method that enables the absolute quantification of nucleic acids. Typical nucleic acid extraction techniques used for qPCR to purify DNA or RNA from unnecessary sample components are compatible with digital PCR. Often dPCR is more robust against inhibitors, giving users more latitude in selecting sample preparation methods. (i.e., cell membranes, proteins, etc.).
Clean nucleic acid samples typically generate the highest quality digital PCR results.
Use a sample extraction method optimized for your sample type and minimize physical debris by centrifugation.
Extraction of DNA and RNA helps to remove potentially interfering components to PCR to maximize dPCR reaction efficiency. Additionally, while dPCR technology is well suited to tolerate some PCR inhibitors, it is not entirely immune. Nucleic acid extraction can help maximize your sample’s quality, minimize inhibitors, and improve your dPCR data quality overall.
We recommend you pipette mix your DNA or RNA sample well and spin it down using a tabletop centrifuge at 10,000xg for 2 minutes before adding your sample to the final reaction.
Selecting your digital PCR MasterMix
A MasterMix is a ready-to-use reagent that has all of the necessary PCR components pre-mixed. The benefit of using a mastermix is that the user typically only needs to supply the input material (DNA or RNA) and the assay (primers and probes) unique to the application.
Several different types of nucleic acid are compatible with digital PCR. The type of nucleic acid used will depend on the researcher’s specific application or experiment.
A few of these nucleic acids include:
- Genomic DNA
- Total RNA or mRNA
- Plasmid or Synthetic DNA
The type of input material used will inform your selection of PCR MasterMix. Combinati offers two MasterMix formulations optimized for usage on Absolute Q. One was designed for use with DNA templates and the other intended for RNA that contains the necessary components for reverse-transcription of RNA to cDNA. These MasterMixes contain a pre-mixed passive reference dye, which the dPCR system uses to assess each partition for proper filling before analysis.
Understanding input material to maximize your digital PCR data
Digital PCR excels in applications dealing with low concentration samples – such as rare target detection. However, it is possible to overload the dPCR reaction with too many molecules.
Avoid overloading the dPCR reaction with too many molecules for the highest accuracy and precision in quantification.
Since Digital PCR measures the concentration of targets by counting the total number of copies present in a reaction, a good to avoid overloading is to estimate the concentration of a sample in terms of copies/microliter (cp/µL).
Obtaining this initial estimate of the concentration of your nucleic acid expected from your sample before starting your experiment is a great first step. There are many online calculators available to help with this.
You will notice the importance of this when determining the number of molecules expected in one nanogram (or picogram, femtogram, etc.) of your sample. This is because genome sizes vary depending on the organism. As seen in the table below, one nanogram of DNA yields a drastically different number of genome copies between humans, bacteria, and flies.
You can apply this same principle to non-genomic sources of DNA, such as synthetic DNA or plasmids. Online calculators can be helpful to estimate the total copies expected from your specific nucleic acid template.
Selecting an appropriate volume of sample
For a typical dPCR reaction of ~20,000 partitions, ensure the volume of the sample you load will result in under 100,000 copies of target per dPCR reaction.
For samples that are low in concentration, filling too many partitions is not a concern. However, when working with a high concentration sample, as can be the case with genomic DNAs or synthetic DNAs, it is possible to overload the dPCR reaction.
During dPCR, the bulk reaction mixture is partitioned into tens of thousands of micro reactions. Digital PCR technology assumes that during partitioning, the distribution of molecules follows a Poisson distribution. Using Poisson statistics, we can determine the concentration of the target in the reaction in copies/microliter without relying on a standard curve.
These calculations are robust enough to provide accurate concentrations even when up to an average of 5 molecules per partition are present in the loaded reaction. Because of this, the upper limit of detection for the dPCR reaction is approximately 5X the number of available partitions. This means that when a reaction is overloaded, the reported concentration becomes inaccurate.
On Absolute Q‘s MAP16 digital PCR consumable, there are 20,480 fixed partitions per dPCR array [right]. This means that in one dPCR array, you can quantify up to 102,400 copies of target per reaction/array.
If your experiment requires a higher upper limit of detection, you can digitally pool up to 16 dPCR arrays of the MAP16 plate to increase the number of partitions for analysis. This improves both the upper (and lower) limits of the dynamic range.
The table below illustrates how the upper limit of quantification is improved as more dPCR arrays are digitally pooled.