I'm starting my PhD on iPSC-derived cardiomyocytes and I'm looking for a reference gene (or papers related to it) that would be suitable to use to evaluate changes in key-proteins expression after a maturation protocol where we expect changes in metabolism (switch towards fatty acid oxydation), structure (bigger, more organised cells), electrophysiology and cell signalling (especially the beta-adrenergic pathway). Any suggestions ?
Have you considered proteomic analysis by MS instead of blots? You can run pathway analysis and you are not restricted to a small set of proteins and internal control.
Dear Camille Charriere
I am not sure of the exact experiment set up but in general GAPDH will work very well for hiPSC-CMs.
You can refer to this publication for more details- Article Catecholamine-Dependent β-Adrenergic Signaling in a Pluripot...
Good luck !
Hello Bill Chi Shun Ho
I have struggled with this very same issue about a month ago. GAPDH worked just fine for me, despite some warnings that cardiomyocytes are very susceptible to changes in metabolism that could interfere with GAPDH expression.
I felt more confident after reading these works (although they approach gene expression within RTqPCR reactions): Article Systematic expression analysis of genes related to generatio...
Article Identification of valid housekeeping genes for quantitative ...
). In fact, for RTqPCR experiments, both GAPDH and HPRT-1 were good reference endogenous controls in my experience.
Don't use GAPDH when you want to study effects on metabolism.
I use for my iPS-CM experiments these instead: 18S, Rpl32, TBP . I find that Actin is not the best reference gene when studying maturation. So avoid that too.
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