multiplex rtqPCR and custom primer/probe design
Posted 22 January 2012 - 04:30 PM
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.
Posted 23 January 2012 - 08:44 AM
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.
I never trust anything that can't be doubted.
Posted 26 February 2012 - 08:40 PM