We are trying to quantify different genes involved in ER stress and autophagy in glioma cells by semiquantitative real-time PCR. The cells are treated with tunicamycin or thapsigargin or are deprived of glucose or glutamine. After 24h, RNA was isolated and the first gene we looked at was ATF4. So far we tested 18S, ß-Actin, RPLP0, RPL13A, and EMC7 as endogenous controls ("housekeepers"). However, all of them are strongly co-regulated by treatment, delta ct-values are around 5. Has anyone faced a similar problem or could suggest alternative controls? Thanks very much for your help.
I disagree the opinion to recommend GAPDH, since ER stress is likely to change the expression of GAPDH in terms of the influence on metabolism.
That is why I strongly recommend the following genes as control.
beta2 microglobulin
beta tubulin
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