Primer efficiency test - (Jan/13/2010 )
I've developed new primers and now, I like to use them in QPCR.
But I should first test the efficiency of my primers. But how do I do that?
Do I just run a QPCR with my primers in different concentrations and my
cDNA in different concentrations and can I then calculate the efficiency? and how then?
Yes; to determine the efficiency of your primer set, run a standard curve (in duplicate or even triplicate). The slope of your standard curve (Ct vs. concentration) is related to the efficiency, and should be as close to -3.321928 as possible.
Efficiency = 10^(-1/slope)
10^(-1/-3.321928) = 2 = exact doubling for each cycle = 100% efficiency
The "standard curve" could be anything containing your target gene: plasmid with target inserted, cDNA from tissues, etc. I typically use cDNA derived from tissue. In this case, the concentration might be unknown, so I use 1/(dilution factor) as a concentration. For example, I make a 1/10, 1/50, 1/250, 1/1250, 1/6250, and 1/31250 (thus a -5X serial dilution, but you could do -10X dilutions instead, if you need a larger range) and I arbitrarily assign the first dilution a value of 10 and the rest of them get a -5X value of that (so then there'd be 10, 2, 0.4, 0.08, 0.016, and 0.0032).
Ideally, the efficiency should be determined for every type of tissue that you're examining, but I've found that the efficiency is usually pretty much the same for different tissues (at least the ones I've examined).
Your primers do not need to be run at different concentrations for the efficiency, just the template.
You should, however, optimize the primer concentration before optimizing your efficiency. To do this, keep the template concentration static and change the primer concentrations. For example, you could run the following sets:
112 nM forward + 112 nM reverse
225 nM F + 225 nM R
450F + 450R
900F + 900R
112F + 225R
112F + 450R
112F + 900R (and the combos of keeping the reverse primer static and changing the F primer concentrations).
The primer concentration set that gives a good Ct (~mid-20's), a high last dRn (i.e. a high fluorescence at the end of the run) and no primer dimers (i.e. a single peak in the dissociation curve), is your best set. That being said, most of the time 300 nM F + 300 nM R is pretty darn good.