RT-PCR consistency - (Dec/13/2007 )
How do you explain the following. A tumor cell line expresses CEA as detected using antibody in FACS and westerns. We are evaluating CEA expression in a panel of tumor cell lines and other cell preps. Using primers for CEA that pick up the molecule in other appropriate controls, we do not get a band in the cell line that was positive by FACS and westerns. So, for some reason, we can't amplify using RT-PCR. Any explanations, copy number I assume? This has been repeatedmultiple times.
although RT-PCR is widely used to study gene expression, RNA levels dont always correlate with protein. This is mostly due to differences in stability of both, proteins usually lasting for longer than RNA. I'll check for RNA expression at a shorter time than you do for protein to asses whether this is the case.
otherwise your RT-PCR protocol needs changing, but i guess from your post that you have controls showing that the reaction does work. How does the RNA from the tumor cells look?
this is a long shot, but another option could be a mutation in the area you are trying to amplify, so the primers don't match enough to allow PCR to work.
maybe try another set of primers.
but first check the RNA and cDNA from the cells.