Multiplex qPCR

What is multiplexing in qPCR?

When qPCR is used to detect multiple target sequences at the same time, this is called multiplex qPCR, or multiplexing. In multiplex qPCR, more than one target gene is amplified in the same reaction, using the same reagent mix. Instead of  running singleplex reactions for each gene, multiplex qPCR allows you to amplify two or more genes in the same reaction.

Multiplex qPCR allows detection of each product in a different fluorescent channel. A benefit of multiplexing is to be able to use multiple probes in one sample. Multiplexing also ensures there is minimal crosstalk between the fluorescent dye/probe used due to similar excitation or emission wavelengths.

As the PCR process amplifies the targeted sequence in a given sample, the level of fluorescence increases with each cycle and is measured and recorded using a qPCR machine, such as the Azure Cielo qPCR System. It is designed for multiplex experiments involving up to six different targets. With the ability to scan 16 wells simultaneously, the Cielo can scan an entire 96-well plate for all six detection channels in just nine seconds. Each sample can be evaluated for multiple pathogens in a minimal amount of time.

The purpose of multiplexing in qPCR

Multiplex qPCR has many applications, from the study of gene expression to genotyping in search of various SNPs connected to disease risk to the rapid detection and differentiation of potential pathogens in a biological sample. With multiplex qPCR, you can use the same reagent mix, but each target region will have its own primer set.

multiplexing in qpcr
Diagram of a multiplex PCR approach
Multiplex qPCR Systems

Designed for multiplex experiments involving up to six different targets

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Traditionally, end-point multiplex PCR analyzes the final gene products by gel electrophoresis. This old method required the final PCR products to be different sizes to be visibly detectable from each other on the gel. Using different fluorescent probes eliminates the need for PCR products to be different sizes; instead of evaluating the size difference visually, the amount of fluorescence is analyzed. Since each PCR product is associated with a specific fluorescent probe, the amount of that fluorescence detected in a sample signifies the target PCR product was present and to what degree.

To successfully use a multiplex PCR approach, a quality qPCR system is critical. The number of samples that can be evaluated is dependent on the different detector channels available in the machine used. For example, if the qPCR system only has 2 different detectors, then only 2 different probes can be used. This limits the number of targets that can be evaluated. For the best multiplex qPCR results, the Cielo is available in both 3- and 6-channel configurations.

Advantages to multiplexing qPCR

Multiplexing offers several advantages (Table 1) for investigators studying more than one target of interest. First, assaying multiple genes per PCR run saves both time and money, providing results more quickly while minimizing consumption of consumables, such as PCR tubes and plates.

Table 1. Advantages of multiplexing qPCR

Benefits of multiplex qPCR
Assaying multiple genes per PCR runSaves on the time it takes to run experiments and analyze results, and money spent on reagents and consumables
Less sample usedThe necessary sample amount can be reduced by measuring the expression of more than one gene
More information is obtained per sampleThis is especially important when you're limited on sample amounts
Improved precisionAmplifying multiple genes in the same wells minimizes pipetting errors

Additionally, more information is obtained per sample, which can be especially important when sample amounts are limited. Lastly, RNA levels or gene copy numbers are compared directly within the same reaction, we do not need controls (to account for pipetting differences between wells or plates).

Best tips for designing and optimizing multiplex qPCR assays

More tips can be found in this app note about multiplexing qPCR.

qPCR Systems capable of Multiplex qPCR

The Azure Cielo is designed for multiplex experiments involving up to six different targets. Innovative optical technology with two sets of 16 optical fibers allows 16 individual wells to be imaged simultaneously, meaning an entire 96-well plate can be imaged in all six fluorescent channels in just nine seconds, saving time and getting results quicker.

Figure 1. Different dyes the Cielo qPCR System is able to detect

Additionally, the Cielo is designed to scan up to 6 individual fluorescent channels that covers most of the qPCR chemistry spectrum (Table 1) allowing you to choose from a variety of PCR probes and ensuring you are not limited to a select few.

Cielo light source illumination
Figure 2. The Cielo is uses a single light source to illuminate each well

This is complemented by an individual-well scanning system, which reduces noise by eliminating the light scatter that can occur when a single light source is used to illuminate a full plate or multiple wells (Figure 2). Additionally, illumination and detection of individual wells improves data consistency across wells and experiments allowing for higher reproducibility.

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