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.
We are having a stability appreciation month, so here are all the good things related to stability from experiment set-up to logistics.
In our last story we talked about how animals can help to improve human health. This time we are talking about how humans could improve the health of animals, more specifically the health of horses and donkeys.
During the last couple of years PCR tests have become quite well-known, mostly in relation to the COVID-19. In reality, using PCR tests to diagnose diseases has been around for some time and it’s becoming increasingly more popular and diverse, allowing for better pathogen detection not just in the field of medicine, but also in other areas such as agriculture.
It is time to introduce another one of our amazing colleagues. For this month we have chosen our development and technology specialist Ilja.