Simultaneous Quantification of Four RNA Targets by Multiplex, Real-Time RT-PCR without Optimization

Researchers carrying out quantitative, multiplex, realtime PCR and RT-PCR require master mixes that provide accurate, simultaneous quantification of multiple targets in the same reaction without time-consuming optimization. Our results at Wyeth Research show that QuantiTect® Multiplex Kits from QIAGEN are well suited for multiplex analysis of up to four RNA targets in two-step and onestep real-time RT-PCR, requiring no PCR optimization and providing the same efficiency as single-target amplification (singleplex) reactions. Multiplex, real-time PCR refers to the use of multiple fluorescently-labeled oligonucleotide probes for the quantification of amplicons produced by different primer pairs in the same reaction. This method is becoming more common with the introduction of cyclers capable of spectrally resolving different fluorophores (e.g., Applied Biosystems® 7500, Mx3000P®, LightCycler® 480, and iCycler iQ®), and with the development of master mixes that facilitate the quantification of multiple targets in the same reaction tube. Some master mixes intended for singleplex analysis have been adapted for multiplex analysis1, 2, but require time-consuming optimization of primer–probe sets. Therefore, evaluating and selecting a suitable master mix is critical when establishing multiplex assays.