I agree with John E. Mindur. I suggest to check that the PCR amplification efficiency for the internal standard (Actin gene) and the target gene match well. At what Ct value do you see the target gene detection ?

Can I also urge you to validate your choice of reference genes and use more than one. `for further information see attached paper.

In relative quantification, you analyze changes in gene expression in a given sample relative to another reference sample (such as an untreated control sample). You can conduct relative quantification when you measure gene expression in response to a drug.

Thank you all for your valuable suggestions.