Hi Krista Mf. How are you doing?

If your endogenous control is working, probably is the target gene primers is not working.

You already increased the cDNA quantity in the reaction, so is almost certanly the primers.

Try to run an conventional PCR and an agarose gel to acess the bands. If you see no bands, it is definitly your primers. Run the GAPDH as a control toghether in this reaction.

Kind regards.

Dear Krista Mf. GAPDH Ct values seem late, that means your samples might be diluted. I would suggest you to try one sample and take 1ug of total RNA per cDNA synthesis reaction (usually 20 ul volume). Then you can dilute the sample up to 5-times, and take 1 ul per real-time PCR reaction. In these conditions I usually get GAPDH Ct earlier than 20. How many cycles did you use in real-time PCR? Did you check amplification target products in melting curve or in gel? The other reasons may be not optimal primers or low expression level of your target gene.

Like Aitsana Maslakova , I agree your GAPDH values are consistent with over-diluted samples.

When you say "cDNA was diluted to 500ng/ul", how did you determine the concentration of cDNA?

I note also that 500ng/ul is _massively_ concentrated by cDNA standards: I use mine at ~4ng/ul and that's fine even for low abundance transcripts.

For reference, dilution of cDNA is a good thing (RT buffer can be inhibitory to PCR, so dilution minimises this) but 1/10 or 1/20 (v:v) is as dilute as you need to go.