RT-PCR and/ or qRT-PCR

What will you suspect when you get a negative expression of your gene of interest but a positive expression of your housekeeping gene? Furthermore, maybe you wish to try to get a hint if your primer set is working fine by amplifying the gene of interest with a DNA sample instead of a RNA sample, and yes, it amplifies.......

So, what could go wrong here?

• Poor RNA quality? May be. But should be checked prior to any downstream assay, if possible.
• Not optimized primer design? Could be.
• Not optimized RT-PCR or qRT-PCR condition? Perhaps. I still remember when I used to amplify low abundant gene (s), and just by increasing the number of cycle from 40 to 50 cycles, all the genes got amplified.

Or the gene is not expressed in the sample then...... Worth to get this investigated, I guess, in my opinion.