I tested gene expression by RT PCR followed by Western blotting to test protein expression. I get an inverse correlation with up-regulation at mRNA level and down-regulation at the protein level. What could be the reason. Please suggest.
Here are some suggestions, why this may be the case:
https://www.researchgate.net/post/What_could_be_the_possible_explaination_of_protein_upregulation_while_mRNA_downregulation
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731011/
If there is no technical error, your result is normal.
You cannot make a correlation between the quantity of mRNA and the proteins produced.
The transcribed mRNAs are not automatically translated into proteins, there is what we call translational regulation and post-translational regulation, and these are 2 regulations which allow us to have a functional protein afterwards.
So even if an mRNA is present, it is not automatically translated into proteins.
Few things you should be looking at,
1) Are there any isoforms for your protein of interest? If yes, the antibody you used is specific to isoform or targets all isoforms?
2) Is your designed qPCR primers spanning the exon(s) of your gene of interest are the coding sequences for the isoform of interest you are studying on?
Given that there is no error in western blot, if the amount of target RNA expressed doesn't reciprocate into the protein quantity, it is likely that there is an RNA epigenetic machinery acting on it. You could as well look at the lncRNAs that are anti-sense to your target gene of interest which could interfering with the translation.
I have done this type of experiment many times using qPCR or microarray for RNA and ELISA or mass spec for proteins. I rarely got a positive correlation between RNA and protein levels. Negative feedback mechanisms or protein degradation could explain this situation.
Hi Aishwarya,
It's not very clear from your question whether you are trying to test the expression of an exogenous gene in an expression system or you are testing the expression of an endogenous gene. Also when you are using the term up-regulation, what is the control in your experiment, are you trying to induce the expression of the gene/protein in an exogenous system or looking at the endogenous gene/protein expression in response to certain conditions like stress/chemical etc..
In any case, it's not unusual to have inverse correlation between mRNA and protein. There may be distinct possibilities depending on the nature of the protein and the expression cell/system being used. In case of an endogenous expression, it's possible that your protein has a high turnover rate and perhaps has a short half life. Therefore, while to compensate for the loss of the protein, the cells are producing high amounts of mRNA but on the other hand protein is getting degraded fast. You can try to treat the cells with MG132 (proteasomal inhibitor) for 4 hours before harvesting them for extract preparation and western blot.
If you are testing the expression of an exogenous gene, its possible that your protein is toxic to the exogenous system and hence while mRNA is being produced the protein synthesis is limited or being degraded by the host defense through proteasomal system.
Hope this helps!
I'm assuming you are using samples from the entire organism/tissue and not an expression vector? I'm also going to assume you are studying a eukaryotic organism & not a bacteria or archaea.
It's normal & common to have a "lag" between mRNA abundance and protein abundance. Check the literature for what is already known for your gene of interest.
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