Bio-Rad Laboratories's amplification application note (technical note number 5438), describes a streamlined approach for obtaining optimal results in multiplex qPCR assays using iQ Multiplex Powermix
To be effective, multiplexing requires some initial optimisation of individual reactions.
Once the assays have been validated, multiplexing is a powerful tool that can provide much data from small sample quantities.
Until recently, qPCR multiplexing was accomplished by supplementing a real-time 2x supermix with additional dNTPs, MgCl2 and Taq polymerase so that limiting reagents would not influence any of the reactions.
Another approach is to determine the limiting primer concentration for the more highly expressed targets by testing a series of concentrations.
A more practical approach, however, is to use iQ multiplex powermix, which allows multiplexing of up to five unique targets with minimal need for optimizing reaction conditions providing considerable savings in cost and time.
Supported by numerous graphs, figures, and tables, the four-page technical note provides the context for multiplex quantitative studies of gene expression using qPCR before describing the guidelines for experimental design in great detail.
Technical note number 5438 is available from Bio-rad using the links provided.
