I have a very difficult problem to solve with real-time pcrs: I am using dilutions of cDNA pools to test for primer efficiency. The same day I make new dilutions (1:10, 1:50, 1:100,..) I obtain a good standard curve; when I freeze the dilutions and I repeat the same reactions the day after I obtain higher cycles for each dilutions, and they increase if I freeze again my dilutions and repeat the reaction for a third time. It seems that more time passes more my cDNA degrades. The least diluted cDNA (1:10) is the one that is more stable over repetitions.
I use DNase-free RNase-free water and filter tips. The negative control is clean.
How can I know if I have contamination in my original RNA or cDNA?
Please if you have any suggestions they would be of great help for me!
Thank you very much!