I would like to target multiple genes in a single experiment (multiplexing) using siRNA. What do I need to know about this before I start?

We suggest using predesigned siRNAs for the different genes. We have a application note showing that we could successfully silence three different human AKT genes, AKT1, 2 and 3 in a single experiment (link: http://dharmacon.horizondiscovery.com/uploadedfiles/resources/app-note-detection-of-related-akt-protein-kinase-family-members-using-solaris-qpcr-assays.pdf). Please see the methods section of this application note for the protocol. Under optimum transfection conditions, transfection of different gene specific siRNAs should result in comparable knockdown to when each siRNA is transfected independently. However, keep in mind that different siRNA duplexes may have different efficiencies with respect to the cellular silencing mechanism, so relative siRNA concentrations may need to be optimized for your system. The total number of gene targets that can be silenced in a single trial will depend on a number of experimental factors including siRNA function, the target gene pathway(s), and the cellular response to elimination of that pathway.