I performed real-time PCR on luteal cells from cows which were treated with various factors for a period of 6 hours. While for western blot, cells were stimulated with the same factors but for a period of 24 hours. Is it possible that a given factor inhibits the expression of the studied gene and increases their protein level? Is the opposite situation also possible, when there is an increase in gene expression, while in western blots a decreased level of protein is observed? Is the difference in times of influence of the given factor could be the main reason or or other mechanisms may cause such results?
Hi,
It is important that you use good housekeeping genes for your model organism as a control for both WB and qRT-PCR. If your results show opposite accumulation of mRNA and protein of your target gene maybe you have posttrancriptional controls regulating RNA stability, translation efficiency or protein degradation. Best wishes.
Hi,
It is important that you use good housekeeping genes for your model organism as a control for both WB and qRT-PCR. If your results show opposite accumulation of mRNA and protein of your target gene maybe you have posttrancriptional controls regulating RNA stability, translation efficiency or protein degradation. Best wishes.
Hey Robert,
happy new year to you.
in many cases mRNA levels correlate with protein levels, but this might not always be the case. RNA biology research gets a big momentum and mRNA can be stored or faster degraded in demand of the encoded protein. So mRNA present or absent in the cells does not necessarily mean that the protein level correlates to it.
We made these experience to times in macrophages. One time, mRNA levels for Cd11b, ASMase and Nox2 were equal between two macropage groups, we investigated. But protein levels showed a completely different pattern. Western Blot showed that peritoneal macrophages had much more CD11b, ASMase and Nox2 than bone marrow derived macrophages. So here, while the mRNA tells you all is equal, the functional protein levels in the cell are not.
For the example please have a look at:
Article The β2 Integrin Mac-1 Induces Protective LC3-Associated Phag...
The other example was observed in microglia. Here the mRNA of angiogenic growth factors VEGF, Ang1 and Ang2 was increased after stimulation, but only VEGF was produced and secreted as actual protein.
For that have a look at:
Article The TSPO-NOX1 axis controls phagocyte-triggered pathological...
So in my opinion, only mRNA levels will not tell you the whole story.
All the best and stay healthy,
Marc
No, I do not think that this is the case.
In my opinion, this is a rather artificial observation due to methodological problems/issues. Maybe, used antibodies or oligors (PCR conditions) are rather inadequate or unspecific, contols and/or the choosen housekeeping factors are not well suited and/or the experimentator is a source for uncertainties regarding experimentation and data nalyses.
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