I'm currently analyzing RT-qPCR data and trying to use efficiency-corrected ddCt method (Fold change=E^ddCt) between 10 very different samples. My problem is that the starting template numbers are very small, resulting in Cq values often >30. In order to figure out efficiency slope, you need at least 3 data points of 10-fold dilutions. Since I ran my samples for 40 cycles, this makes calculating efficiency slopes impossible for some of the samples with the lowest starting quantities.
So my question is, if I have some samples with acceptable efficiency slopes but others without, is it still appropriate to use this method? I would have to assume that the ones without a calculated slope to have efficiencies of 2 or 100%, which is not likely true, and I'm afraid of biasing my data.
Has anyone else had this problem or had any thoughts? Thanks!