Comparing SYBR and probe-based Ct values - Is it valid? (Mar/19/2011 )
I have an assay, where I used UPL probe (Taqman probe based system) for one of the amplicons and SYBR green I based system for other (we wanted both to be TaqMan-based to increase specificity, but we couldn't design the second amplicon using UPL due to the high sequence homology).
Now, I have Cts and efficiency (from dilution series) from probe-based and from SYBR-based system and I want to use then in the same equation (Pfaffl, efficiency corrected).
I can't find any logical reason why this wouldn't be valid, I mean when you got Ct and efficiency, there should be no problem acquiring it from different methods (I think the same stands for 2nd derivative vs fit-points methods as long as you calculate Cts and efficiency same way). Or is there something obvious I'm missing? Could someone rightfully object about using different methods for each gene?
Thanks for any comments.
I feel like there might be a few differences between the calculations, but not sure how big of an effect they might have on the overall outcome. B/c the probe based system is only going to give off a signal to the qPCR machine when the proper sequence is amplified, so you know the amplification is correct. However, with the SYBR system, any DNA amplification is going to give off a signal so if you get any non-specific amplification that is going to be included in the final Ct reading, etc. I feel tho, that if your melt peaks are really clean on the SYBR run, then you can pretty confidently say that you are amplifying the right signal and thus the two different methods are comparable.
Good point about SYBR, there has to be a single distinct peak. Of course in my case there is single specific peak on the melting.