multiplex rtqPCR and custom primer/probe design - (Jan/22/2012 )
I've been asked to design rtqPCRs for four cytokine assays in koalas (limited sequence available). I was wondering what the general feeling on multiplexing qPCRs is these days - particularly up to four sets + house keeping gene?
Secondly, does anyone have any experience with custom made primer/probe sets? We are on a limited time frame and were wondering if getting these designed will save us time.
Five-plex? Nope. I wouldn't do any more than duplex for quantification, and well.. I wouldn't do even the duplex if I'm not limited by the template. Too much optimizing. First there is problem with fluorophores, machines have dozens of different filters, but the windows are overlaping, there is spectral crosstalk, usually solved well in the common FAM/VIC duplex, but I wouldn't want to see it with three other. Also other fluorophores other than VIC can have notably lower intensity, that can be problematic. And are more expensive.
And different thing is the multiplex PCR itself, IMO there is so much interference, that I wouldn't trust that quantification much. In duplex you can limit the primers in the more intense reaction (usually housekeeping) but how you can do it with other unpredictible quantities I don't know.
Custom made primer/probes is now the way to go. Our machine's company (Roche) offered us an option of free design by it's probe synthesis company, and small test batch to check for real, before we payed for that. We don't design Taqman or melting probes ourselves anymore, we just write them.
There are now databases of Taqman assays both by ABI and Roche (and others for sure), but I would bet they don't have koalas there.
Thanks so much for this answer. I thought as much re the multiplex, but this is a nice answer to show to my supervisor. Rumour has it the koala genome is coming....