Obviously I have heard about the discussions about the suitability of GAPDH as a control gene for qPCR. But since everyone, so it seems, keeps using it, I didn't bother to change anything. Now, running GAPDH side by side with an Actin control I noticed a remarkable downregulation of GAPDH-expression after treatment induced cell stress (X-ray, Oxaliplatin, Doxorubicin). This result I got independently in several experiments. Since, to my knowledge, the current literature doesn't cover this subject very much, I would love to hear about your experiences and knowledge.
In the past we have seen changes in GAPDH expression as well after exposure of cells to genotoxic agents. Therefore, we always use multiple 'house-keeping' genes in our qPCR to be able to exclude unexpected effects of the exposure. We also routinely check expression of the control gene by comparing the Ct-value in the exposed group vs. the unexposed group, assuming equal amounts of cDNA.
Excellent, this is exactly what I thought to do and confirms what I see. Its just surprising that so many researchers, including myself, are using just one standard housekeeping gene and assume the data are always solid. Thanks a lot for your answer Roger.
Sven,
For your information:
Bustin, Stephen A., et al. "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments." Clinical chemistry 55.4 (2009): 611-622.
The MIQE guidelines state:
“Normalization involves reporting the ratios of the mRNA concentrations of the genes of interest to those of the reference genes. Reference gene mRNAs should be stably expressed, and their abundances should show strong correlation with the total amounts of mRNA present in the samples.
Normalization against a single reference gene is not acceptable unless the investigators present clear evidence for the reviewers that confirms its invariant expression under the experimental conditions described.”
Since, GAPDH is an enzyme in Glycolysis pathway, any treatment effecting the energy metabolism is expected to alter the GAPDH expression. For example, it is not a suitable reference gene for normalization qRT-PCR data in spermatozoa for study the effect of cryopreservation.
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