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Current best practices for gene expression analysis by qPCR recommend the use of multiple normalizers and, potentially, a second gene of interest (GOI) assay. For samples with limited nucleic acid content, such an experiment may only be achieved by performing multiplexed qPCR assays. Even when working with abundant nucleic acids, multiplexing can save time and money. Achieving successful multiplex assays depends upon several criteria, including using robust assays, correct reagent and target concentrations, and appropriate cycling parameters. Using data from several assays, we demonstrate how PrimeTime™ qPCR Assays simplify a 4-plex assay system, and discuss factors that influence the success of using these assays in a multiplex format. We also compare the use of master mixes assembled de novo to commercially available reagents and discuss their effects on multiplex assays.