There could be several reasons for not getting any Ct value for your genes of interest in H9c2 cells. Here are some possible reasons and suggestions to troubleshoot:

In addition, you can try running a positive control with RNA or cDNA from a different rat tissue or cell line to confirm that your primers are working. You can also try running a negative control with no template to confirm that there is no contamination in your reaction. If you have tried all of these troubleshooting steps and are still not getting any Ct values for your genes of interest, it may be worth considering alternative techniques such as in situ hybridization or protein expression analysis to confirm gene expression in H9c2 cells.

Samiullah Khan Thanks for the detailed explanation to answer my issue. I have already troubleshot the above reasons. I have checked these primers with rat cell lines and rat tissues where I am getting lower Ct values of GAPDH and b-actin genes compared to H9c2 cells. I am getting a high Ct value of these housekeeping genes and no Ct value in other primers with good Ct in other Rat cell lines and rat tissues. Primers and cDNA are fine but something is weird with H9c2 cells. Also, I have checked expression in the early passage of H9c2 cells but the results are the same. I think you are right I should go with protein expression.

Thanks a lot.

You're welcome! It's always frustrating when something seems weird or unexplainable. Keep up the good work

Thanks for the wishes. Samiullah Khan