I ran the same qPCR test (SYBR Green) on a lot of samples that didn't fit on one plate (over 20 plates all together) and want to compare all of them to the same standard curve that was included in one of those plates. I did have a few positive controls on each plate to check variability between plates.
Now I am aware that I need to set parameters in the same way for all the plates in order to compare them. The threshold will be a fixed value, but I am wondering about the baseline. Ideally, it would be fixed at a certain intensity for all plates. That is not possible in the software I'm using (Eppendorf MasterCycler ep realplex, v. 2.2), so I can either use automatic baseline or manual baseline with specifying start and end cycle.
In a few cases, automatic baseline gives me much better results than manual baseline (with manual setting the curves are not parallel, no matter which cycle range I choose provided I stay below the first amplification; while with automatic baseline the curves are nicely parallel).
I think automatic baseline is not a good option for comparison, but I'm still wondering what is the best thing to do. Even if I choose manual baseline and specify start and end cycle, I think it won't be exactly comparable between plates, but that's probably the best option, just to keep in mind that some results might be of a slightly lower quality because of that.
Any thoughts?