Roche second derivative maximum - can be treated according to Pfaffl? (Feb/01/2011 )
I've been using Roche's Lightcycler 480 for qPCR and determining Cp using second derivative maximum, instead of fit-points. The thing is I don't do the analysis in Roche's software. I take it to excel and treat it there using Pfaffl's method. Lately we've been wondering if this is correct, since Pfaffl method is used for fit-points.
Does anyone have any advice on this? From what I've seen I think the two methods are equivalent, but I don't know enough math and kinetics and all to be 100% sure about this.
Thanks in advance!
I have never used regulary fit-points method on LC480, because it just seemed to me less accurate. I use derivative maximum in relative quant. software and in my own calculations in Excel (with High confidence setting, not the other!). Do you have some citation from Pfaffl paper, that the formula should only be used for a fit-point method? I'm pretty sure, that it doesn't really matter which method you use to calculate Cts, since you calculate them same way for all your samples/standards. The Cts may differ between methods, but the results should be the same.
Personaly I think the derivative maximum is better than fit-points, and the machine have fit-points only for those used to that kind of analysis. I only use fit-points when there are some troubles with the amplification curve, if it's not flat on the beginning or have "zig-zag" in the region where it slowly starts to increase (happens sometimes when using same plate for more than two runs). In that situation is der-max highly unreliable, but you will notice that in the graph. And in that case it is of course better to repeat whole reaction.