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When interrogating 3 or 4 targets in the same well, there is even more competition for shared reagents than in a duplex reaction, and the scope for unwanted interactions between primers and probes increases. This can provide huge savings in cost, reagents and time, but the resulting experiments are more complex, and validation becomes more time-consuming. You can, under carefully optimized conditions, perform multiplex qPCR to measure the expression of three or four genes simultaneously in a reaction. The emission spectra for FAM and VIC peak at 517nm (in the blue region of the visible spectrum) and 551nm (in the green region) respectively (Figure 1), making these wavelengths easily distinguishable by any Applied Biosystems real time PCR instrument. Typically, the probe for your gene of interest is labeled with FAM dye and the probe for the control with VIC dye. The real time PCR instrument used for performing amplification must be capable of distinguishing precisely between these fluorescent labels and measuring the signals produced by the amplification of each gene.
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TaqMan assays to detect the target and control genes, containing probes that have been labeled with 2 distinct fluorescent dyes, are added to the same reaction, and use the same pool of Taq polymerase enzymes, nucleotides, and other reagents during amplification. Typically, in a relative qPCR experiment designed to determine the fold change differences in gene expression, these will be a single gene of interest (target gene) and an endogenous control. The simplest, and most commonly used, type of multiplexing is duplexing, in which two genes are amplified in a single reaction.