threshold for real time PCR - (Apr/01/2005 )
Hi, I am using iCycler for real time PCR. I do standard curve (1/5 to 1/625 dilutions) for each gene. My questions are:
1. Do I have to use same threshold for both GAPDH and my target gene?
2. May I change baseline cycle? or do I have to use auto calculation?
3. When I used "PCR baseline substracted curve fit" and auto calculated threshold, my GAPDH primer PCR efficiency was around 70%. But When change the baseline cycles 2-10 (user defined), it's PCR efficiency increased to 92%. Is that okey?
4. If I use user defined baseline for GAPDH, do I have to use same values for my target gene?
Thanks a lot.
Hi! I'll try to answer you questions:
1. You have to use the same treshold. This is a way to normailize between the interal control and the genes. The curve doesn't start at the same point for all the genes. You have to use a treshold where all the genes are in the exponential phase and you are not cutting in the background fluorescence.
2. Depending on you Ct values, you have to change your baseline. For instance, with TaqMan technology of Applied Biosystems, the say that if you genes appear before cycle 15 you have to adjust the baseline. You can get more information in the web of Applied Biosystems, in their guidelines to do Real Time.
3.I think it is OK. Please, again, check at which cycles appear your genes and the guidelines I have told you before.
4.Yes, you have to use the same baseline.