When I export the data from real-time to the software copycaller some plots are between 1 and 2 numbers of copies, how do I know if the sample contains 1 or 2 copies of my gene of interest?
I had the same situation as yours. In most of cases, I didn't base on CopyCaller. I only used its mathematical formula to calculate copy number. I also used some conrtol cases with copy numbers known as 1. So, based on the values from the control cases, I can know which sample contains 1 or 2 copies of my gene.
droplet digital PCR (ddPCR) may be something to try since you are dealing with such small copy numbers. The resolution of qPCR/RT-qPCR may not be on your side here...
Thanks for helping me. But I used a control case with copy numbers known as 2. Do you think I might be wrong? Maybe if I use a control with copy numbers known as 1 it will be easier?
I think you were not wrong. You can check two following factors.
1. The quality of your sample. You can check it by looking at Ct SD [One Plus PCR machine] or sigma (delta Ct) [CopyCaller]. If it is lower than 0.15, you may not trust that copy number.
2. The stability of your reference gene. As usual, the Ct value of reference gene will not be changed too much in the same plate. Treating Ct value of reference gene on your calibrator sample as a standard value, if Ct value of reference gene on your sample differs standard from at least 1 cycle, that means your reference gene may have some problems. Then, you can try another reference gene.
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