Hi 

I have been using the same primers for a year and a half to amplify my gene of interest using QRT-PCR but now when I run the QRT, I am not getting a signal for this particular gene. I do not have a problem for my other primers, in which I freeze-thaw the different primers at the same rate. I have tried multiple different scenarios to determine what is happening with my primers. 

1)I have order new primers using the same sequence, so that I can rule out the degradation issue with the old primers.

2)I have ran the conventional PCR using my old and new primers with cDNA to rule out the design issue of the primers. In this case, I got bands with cDNA but not with my genomic DNA. 

3) I have also used old cDNA and new cDNA to determine if there was an issue with my cDNA

4) I have also ran a RNA gel to determine the quality of my RNA.

Has anyone had similar issues with their primers? I am totally confused as to what is actually happening because these primers used to work. 

Any suggestions?

Thanks!