Solving Multiplexing Issues
Multiplex, real-time PCR enables the amplification of two or more targets in a single reaction. “It’s increasingly being accepted as the technology of choice when needing to simultaneously analyze expression of one or more genes of interest with reference genes as well as for pathogen detection,” said Mark Richards, marketing manager at Qiagen.
Richards noted that researchers still face many challenges when trying to optimize multiplex qPCR. “Multiplexing can be tedious and time consuming because of the need to establish optimization procedures such as adjusting enzyme, primer, DNA, and magnesium concentrations in order to provide the best signal-to-noise ratio. Even after this extensive work, many cases still prove disappointing.”
Qiagen has developed a number of technologies for multiplex PCR that eliminate the need for optimization. “The multiplex PCR technology uses a highly stringent hot-start enzyme, an ammonium containing buffer, and a multiplex PCR-enhancing synthetic factor MP that eliminates the need for optimization because it supports macromolecular crowding. That is, it allows efficient annealing of multiple primers under identical cycling conditions. Scientists have shown that they can detect up to seven targets with this technology.”
“With this system, primer concentrations do not have to be optimized. One can use the same concentration of each. Of course, the primers must be suitable and optimized for the sequence, just as in any singleplex assay,” Annette Tietze, Ph.D., senior global product manager for qPCR, added.