Practical evaluation of universal conditions for four-plex quantitative PCR.

Multiplexing quantitative polymerase chain reaction (qPCR) is a powerful way to substantially increase the number of genes that can be analyzed, while also reducing sample requirements, time, and cost. However, little previous work has been done to show its feasibility for multiple gene targets. Here, we determined optimal conditions for four-color multiplex qPCR. On the basis of amplification curves, we first established that the concentration of probe-primers should be about tenfold lower than that for conventional qPCR. This condition was evaluated using four sets of probe-primers labeled with FAM, CAL Fluor Orange, TAMRA, and Quasar670, respectively. To simulate the condition that different genes have different levels of transcript abundance, a series of test samples was prepared by mixing a constant amount of two kinds of vector together with different amounts of two other vectors in a four-plex qPCR format. The PCR efficiency of the constant genes was minimally affected by the presence of the spiked vectors, and the slope factors of standard curves for the two spiked genes were sufficient for the accurate quantification. We demonstrated here that qPCR in a four-plexed format is feasible for cost-effective practical use through a combination of lower concentrations of probe-primers, an appropriate reagent, and a detection instrument.