I have been trying to check the expression of a pro-inflammatory cytokine between differentiated wild-type and knocked-down THP-1 macrophages cells. When I stimulate the cells (both at 24h and 6h)mRNA expression is reduced in kd-THP1 cells (by qPCR), but not the protein (checked by ELISA). The protein levels are similar between both the cells, when I have significant reduction in the mRNA levels. My question is, if anyone faced the same issue or any way that I can overcome this issue?
This is not an issue at all and very common in secretome analysis. What matters for you is that both ELISA and western blot goes parallel with each other.
The explanation would be that your gene of interest is subjected to post transcriptional modifications, and that's normal and common (which is the case IL-6 for example)
same problem here, but in different fashion,
My ELISA results indicates my sample dose-dependently reduce IL-6 in RAW 264.7 cells but my qPCR result indicates dose-dependent increase.
3h stim for PCR
24h stim for ELISA.
@Donghan Kim
By 3hr stim, you mean 3 hours stimulation with your drug/reagent?
If this is the case, you can't jump to any conclusion before comparing them both at exact same stimulation time. Meaning that you have to do ELISA at 3hr and qPCR after 24 hrs for the conditions you indicated above.
After doing these experiments and incase you have the same results (decrease gene expression and increase in protein expression), your drug/reagent is maybe inducing post transcriptional/translational modofications on IL-6 since as I mentioned above it's one of the gene that are very prone to this kind of modifications (depending what is the type of post transcriptional modification, it could yeild either increase or decrease in protein expression, depending on the context?) .
But, again, you can't jump to a comclusion before assessing mRNA and protein at the same time interval.
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