Optimizing multiplex RT-qPCR - (Jan/26/2009 )

I have done only single-plex RT-qPCR in the past and now am attempting to do multiplex. I have turned to a variety of resources for help on optimizing the mRT-qPCR process, and there seems to be a few different ideas on how to do this. After you test your linear range of amplification (by a dilution series of template) for your primer pair to ensure equal amp. efficiency:

1) Use equivalent amounts of primers and adjust thermocycling settings (ie annealing, extension etc) and also amounts of other components (ie dNTP, enzyme, buffer)

OR

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?

adding the same amounts of primers/probes can make them to compete and if you have a gene that express a lot and the other not you may not have both amplification. What I do for example is I trite and use the least concentration possible for the housekeeping gene.

If you look in my real time PCR documents thread you will find the "ABI user bulletin_5" which extensively explains how to optimize a multiplex real time PCR reaction.

So, for all those interested, I am going to be using a mastermix specifically for multiplexing, which will (purportedly) ..."...accurately quantify both low and high-abundance targets in the same tube... regardless of rare or limited sample size." Therefore, the steps I need to follow for optimizing the reaction are to:

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