Optimizing multiplex RT-qPCR
Posted 26 January 2009 - 11:47 AM
1) Use equivalent amounts of primers and adjust thermocycling settings (ie annealing, extension etc) and also amounts of other components (ie dNTP, enzyme, buffer)
2) Find optimum concentration of primer and probe by evaluating best Ct vs dRn
Now, I have no real idea why you would start with one or the other, and how you would incorporate both possibilities for adjustment into your optimization process,, so I guess I want to know what I should do after I've got my primers in hand, or HOW DO YOU OPTIMIZE YOUR mRT-qPCR?
Posted 27 January 2009 - 12:40 PM
I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Posted 29 January 2009 - 10:09 AM
Posted 02 February 2009 - 07:38 AM
1) Make sure I get similar amplification efficiency of each primer pair by doing a SYBR green chemistry over my needed linear dynamic range (melt point analysis, std curve, run product on high res gel)
2) Test each primer pair with different annealing temps to find the optimal cycling conditions
3a) Multiplex with probes, adding each primer pair together sequentially to make sure there is no cross reactivity
3b) troubleshoot if cross-reactivity occurs by adjusting reaction cycle parameters based on the problem encountered (i.e. if short non-specific product, try: increasing annealing time, temp, and extension temp; decreasing KCl buffer concentration etc FrommPCR troubleshooting website