There are two types of people out there. The ones that stuff the dishwasher full to the brim. And the ones that won’t hesitate to run it with only one plate inside.
Chances are, if you’re a stuffer, you’re also a multiplexer. You want to do more in one go and save time and resources while at it. But as you try to ram that dishwasher door shut, you know very well that you can easily overdo it. Some of those dishes can come out just as dirty as when they went in.
You might get the same anxious feeling that you might be pushing it too far with PCR multiplexing. The more targets you analyze at once, the more info you get from one undivided sample while using less time and fewer resources. But how far can you go before you get in a tangled mess? Not very far with qPCR. Farther with dPCR multiplexing.
Earlier, we would have said that you can easily multiplex up to five targets with dPCR. Today, we’ve more than doubled that to 12 targets. Double the dishes in the same dishwasher? Yes, please.
To help you analyze 12 targets without having to purchase a new dPCR system, we’ve had to go above and beyond “normal” color multiplexing into high-order multiplexing or amplitude multiplexing. This is where things get interesting.
What is amplitude multiplexing?
Unlike color multiplexing, where targets are detected based on separate fluorescent signals in distinct channels, amplitude-based multiplexing allows multiple targets to be distinguished within the same optical channel. This is achieved by adjusting the concentration of probes, which results in different signal intensities1. In a 1D scatterplot, each partition in a dPCR reaction can exist in one of four states:
- Negative for both targets
- Positive for one target only
- Positive for second target only
- Positive for both targets
By using different probe concentrations, distinct clusters of fluorescence intensity are created, allowing the software to differentiate between multiple targets within the same channel.
Figure 1. How amplitude multiplexing looks like on a QIAcuity Nanoplate signalmap (right) and on a 1D scatterplot (left). Target A has a lower probe concentration (bottom blue band), target B has a higher probe concentration (middle blue band). A mixture of both targets A and B has the highest probe concentration (top blue band).
Tips for getting started with high-order multiplexing
How can you set up 12-plex dPCR assays? Why, you don’t even need 12 tips for that...
- Take good care of your probes. Select probe concentrations and fluorescence intensities that generate distinct signal clusters. Higher probe concentrations lead to stronger fluorescence signals, while lower concentrations create dimmer signals.
- Select your fluorescent dyes carefully – Choose dyes that minimize spectral overlap. If using Long Stokes-Shift (LSS) dyes, make sure you read point 5.
- Use the right chemistry – Benefit from specialized multiplex mixes, such as the QIAcuity High Multiplex Probe PCR Kit, which is specifically designed for high-order multiplexing. Thanks to enhanced buffer chemistry and optimal polymerase concentrations, you achieve the sensitivity and robustness needed to resolve complex multiplex reactions.
- Validate singleplex reactions before multiplexing—Test each assay separately before combining them into a multiplex reaction. This step confirms that each probe functions correctly and lowers the chances of multiplex mishaps.
- Apply custom cross talk compensation – Optical cross talk occurs when fluorescent signals bleed into neighboring channels, potentially leading to false positives. The QIAcuity Digital PCR System offers a new software feature for creating a custom cross talk matrix (CXTM), which accounts for different dye interactions and improves signal clarity.
- Exercise caution when setting thresholds – With the QIAcuity software, you can set multiple threshold settings per channel to classify your partitions with higher accuracy. Manually adjust these thresholds to refine data interpretation and confirm the fluorescence intensity clusters are well-separated.
Interested in learning more about amplitude multiplexing? Watch a quick tutorial video on how to set up multiplex assays and analyze the data.
References
- Whale AS, Huggett JF, Tzonev S. Fundamentals of multiplexing with digital PCR. Biomol Detect Quantif. 2016;10:15–23.