You got yourself a smartwatch. A quick (or no) skim of the instructions manual and you can already tell the date and time. Dig a bit deeper and suddenly you can monitor your heart rate, find your way home, pay at the grocery store and send emails.
The same goes for your digital PCR system.
Get one and you can pretty much immediately start quantifying DNA and RNA with absolute results and higher sensitivity than qPCR. Invest a bit of time exploring and suddenly you can do fancy things like 2-D scatterplots, multiplexing and assay optimization.
Our newest course, “Learn and Grow 2.0: A dPCR deep dive,” is both your quick and advanced instructions manual. We’ve pulled six digital PCR tips from there for beginners and more experienced users.
Beginner dPCR tips
Tip 1: Don’t skip the dilution step
If your sample concentration is too high, partitions can contain multiple target molecules, complicating the analysis. Start with a dilution series to ensure that the DNA is evenly and randomly distributed, giving you clean “yes/no” signals.
Tip 2: Define your sensitivity limits
For high-quality experiments, determine your assay’s Limit of Blank (LOB), Limit of Detection (LOD) and Limit of Quantification (LOQ). These parameters help you distinguish between background noise, true positives and results that can be quantified with accuracy.
Tip 3: Use reference genes for gene expression analysis
Even though dPCR offers absolute quantification and it’s much easier to multiplex, it still requires reference genes if you’re studying mRNA or microRNAs. This is because you should normalize your gene of interest against stable reference genes (like GAPDH, TBP, or RPL13A). This corrects for differences in RNA input and ensures reliable results.
Advanced dPCR tips
Tip 1: Minimize rain
A lot of rain, or the cloud of partitions between your positive and negative partitions, indicates that you need to optimize your assay. You can do this in four ways:
- Adjust annealing temperature
- Try different cycling conditions
- Change primer-probe concentrations
- Use annealing gradients
Tip 2: Save sample and resources with higher-order multiplexing
Leverage amplitude multiplexing to simultaneously quantify two targets in the same color channel to scale up your experiments. This approach on the QIAcuity allows detection of up to 12 targets in one reaction, saving precious samples and resources.
Tip 3: Consider one-step RT-dPCR vs two-step RT-dPCR
One-step RT-dPCR is when reverse transcription (RT) and PCR amplification occur in the same tube. Two-step RT-dPCR occurs when RNA is converted to cDNA in a dedicated RT reaction and then the cDNA is aliquoted into dPCR reactions. You can pick an approach that suits your needs better. Bear in mind that:
- One-step RT-dPCR is faster, cheaper, and simpler for routine analysis
- Two-step RT-dPCR allows more flexibility, like targeting microRNAs or siRNA-cleaved mRNAs