Thermo Fisher Scientific, Vantaa, Finland
Multiplex reactions offer several advantages over singleplex reactions. Combining several targets into the same reaction brings substantial time and reagent savings. Furthermore the detection of more than one sequence within the same reaction vessel allows control reactions to be included. Internal controls can be used to detect reaction inhibition, which may cause false negative results.
Optimizing multiplex reactions brings several challenges due to the complex interaction of several primers and probes. Amplicons compete for the available reagents, such as primers, polymerase and nucleotides. Other issues include designing of primers in a way to avoid primer dimer formation and the optimization of primer concentrations to enable several targets to be amplified in the same reaction.
Multiplexing qPCR reaction also requires careful choice of fluorescent dyes and matching quenchers. Choosing the correct dyes to fit qPCR instruments may be difficult, because all instruments contain unique excitation wavelengths and filters for both excitation and emission. However, most instruments come with a pre-calibrated dye set and usually with the possibility to calibrate additional dyes.
In publications, laborious process of optimizing multiplex reactions is often performed with a single instrument and reagent. Replicating the results with a different instrumentation and reagents often results in varying degree of success. Aside from the differences between instrument detection channel wavelengths, not much is known about the transfer of multiplex assays from one instrument to another, representing an interesting topic to study.
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