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The selected fluorophores must also be compatible with the excitation and emission filters of your real-time PCR detection system. To distinguish each reaction, choose reporter fluorophores with minimally overlapping emission spectra. Multiplex qPCR assays require multiple reporters - one to follow each amplification reaction. Selection of Reporters and Quenchers for Probes It is important to ensure that the different primer and probe sets do not exhibit complementarity to one another because all primers and probes will be present in one reaction. A common challenge for multiplexed qPCR assays is the amplification of targets present in significantly different concentrations.ĭesign all primers to have approximately the same T m (55–60☌), and also design all probes to have approximately the same T m (~5–10☌ higher than that of the primers).

Simply combining all the primers and templates in the same tube is not sufficient because the amplification of one target many influence the amplification of other targets in the same tube. Successful multiplexing of qPCR assays requires careful experimental design and optimization of reaction conditions.

Less sample handling and associated opportunities for laboratory contamination.

Increased laboratory throughput, reducing the overall cost of qPCR assays.

Reduced numbers of false negatives when a control target is amplified within each sample.

Conservation of template DNA, which is important when the amount of starting material is limited.Multiplexing confers the following advantages over singleplex reactions: Currently, it is possible to amplify and quantify as many as five targets in a single qPCR assay, depending on the features of your real-time PCR instrument.

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