We are running qRT-PCR in mouse, pig and monkey. We find that we have very low CT values for multiple genes that we expect will be very high - reference genes (GAPDH, YWAHZ) - and inflammatory genes in model of brain injury in the pig and monkey (CT 25) compared to the mouse (CT 16). Does anyone have any idea of what steps in the preparation of the cDNA would be most vulnerable to species variation? We have validated in house designed primers and everything is being prepared (extractions/RT) in the same way. Using the cDNA undiluted is problematic (and expensive)! Thanks!