Multiplex real-time PCR: A guide to application

In multiplex real-time PCR (qPCR), scientists amplify two or more target genes in the same reaction, using the same reagent mix. Multiplexing allows scientists to overcome limitations for myriad applications.

First and foremost, multiplexing enables scientists to conserve valuable samples used in a quantitative assay, which is especially important for research involving difficult-to-obtain specimens. In a single qPCR experiment, only the gene of interest or the control is amplified in each well. If each assay is performed in triplicate, researchers need to divide a sample into multiple wells to measure gene expression, limiting the number of genes that can be tested when using a sample in short supply. Scientists overcome these limitations by amplifying multiple genes in the same reaction. Additionally, researchers reduce cost and time required to perform qPCR because they use fewer reagents per experiment and can run experiments and analyze results more quickly. Because pipetting errors may also be minimized in one reaction versus several, researchers may also benefit from improved precision when performing multiplex qPCR versus single assays.