I have synthesized cDNA using one microgram RNA template. Then, I diluted it to five times. Then, I did PCR using diluted cDNA with housekeeping gene act2. In PCR, I got nice bands in the gel. After that, I used the same cDNA and the same primers for RT-PCR. But there was no amplification, and the melting curve was also bad. What could be the possible reasons for troubleshooting? Should I dilute cDNA again to 10 times or 100 times? or can anyone suggest a way?