Please help me to find a solution for my problem. I have to quantify the transcription of a gene and for this reason I chose the real-time PCR assay. However, I am confronted to the following thing: although my home gene GAPDH is ok, the gene I try to quantify is not. I get two peaks, sometimes equal and others the one is higher. I suspect that the one of these is my gene and the other my be some isoform.
1. Have you ever been confronted to somethng similar?
2. How can I overcome this problem? I tried to use 2λ of cDNA (500ng/λ) and I got worse results than using 1λ from my stock cDNA. Lower concetrations of cDNA have also given me disappointing results.
3. In addition, I use 40 cycles and my product appears from cycle 34 or later and the LightCycler real time PCR i use stops counting from cycle 35. Can you help me with that? What shall i do?
Any thoughts are welcome cause I do not know what can I do...
Thanks in advanced