I had started with information gathered from a microarray which indicated that treatment with my compound increased expression of a certain gene (MAFB) about 3-fold. When I verified with RT-PCR, I noticed I was consistently getting ddCt's of 2 (approximately 4-fold increase) in each individual experiment (I was having issues with getting consistent dCt's so I can do statistics, but that's another story), and my NTC did not . However, Western blot for MAFB revealed that the protein level was not changing at all. I have always noticed good qualitative correlation between mRNA and protein (that is, directions are the same, even if magnitudes aren't). I also understand that there are some papers out there that note a poor correlation between mRNA and protein expression. However, I did not expect this. Has anyone else ever seen such poor correlation, and what explanations could there be for that? I had previously found another protein whose mRNA was upregulated in RT-PCR to be upregulated in protein expression, so general translational issues don't seem to be a problem.
EDIT: OK, I think I found one possible solution. Papers from PNAS (2006) and Nucleic Acids Research (2012) point to miR-130a as a known translational inhibitor of MAFB that does not immediately induce mRNA degradation through (as opposed to miRNAs that do by utilizing Argonaute). The question then becomes whether miR-130a becomes upregulated in tandem with MAFB upon treatment with my compound. Of course, my PI could then argue that it might not be a question worth considering, as it could take us in too many different directions. What say you? I'm beginning to think a microarray analysis that does not take miRNAs into consideration is sorely lacking in information.
Edited by JDSBlueDevl, 16 November 2015 - 06:33 PM.