To help you get into the multiplexing workflow efficiently, there are a few key points to consider:
Figure 1. SolisFAST® Probe qPCR Mix (ROX) was used in a 4-plex probe-based qPCR with fourteen fold serial dilutions of human gDNA (40 ng –40 pg per reaction, three replicates at each concentration). qPCR was performed on a QuantStudio™ 6 Flex qPCR cycler (Applied BioSystems™) using ROX dye for normalization.
Using regular qPCR mixes in highly multiplexed assays carries a number of risks, such as late Ct values, low efficiency, low sensitivity, and low fluorescence. As regular qPCR mixes do not have a sufficient amount of components for amplifying more than 2 targets simultaneously, you are running the risk of getting false-negative results. Using specifically designed multiplex mixes will greatly increase your success.
For fast cycling speed and up to 5-plex reactions:
For standard cycling speed and up to 4-plex reactions:
By using the right product designed for multiplexing, you can rest assured you will enjoy similar sensitivity to singleplexing with a greatly reduced workload.
Figure 2. HOT FIREPol® Multiplex qPCR Mix (Purple) was used to perform 4-plex or single-plex probe-based qPCR with five tenfold serial dilutions of human cDNA (cDNA concentration ranges from 200 ng to 20 pg per reaction). Reactions were performed with Applied BioSystems™ QuantStudio™ 6 cycler using Purple dye for normalization.
Ribonucleases or RNases are native proteins that can degrade RNA molecules. To mitigate risks we rely on RNase inhibitors, which are invaluable tools to protect and preserve RNA molecules in diverse biotechnological applications.
There are two options for target detection when doing qPCR - either you use dye or probe-based detection. Today we are going to take a deep dive into the world of dye-based qPCR, which was invented during the early nineties by Russell Higuchi [1] and is still used daily around the world.
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