1- Timing of the sampling. By the time you collect supernatant, all mRNA have been already translated and degraded.

2- Some transcripts can be translated more than just once, resulting in more proteins than transcripts.

3- Elisa and/or qPCR don't work properly.

2ndAnswerthatisincreaseinthetranslationoftranscriptspresentpossiblyduetoincreasedexpressionofcertainTFs.

I agree with Korcan.  Typically mRNA abundance peaks earlier than protein abundance due to the lag time between transcription and translation. 

Cytokine mRNAs are typically extremely labile due to the presence of AU-rich elements in their 3'UTRs and and as such are degraded quickly to keep the inflammatory response in check.  

I have not studied IL-12b p40 but the majority of cytokine mRNAs contain AU-Rich elements so I would not be surprised if it did as well. 

The answer may also depend on the cells you're culturing/assessing; if this is a culture of bulk splenocytes, you could detect high levels of secreted cytokine but the mRNA derived from the total population and not just the IL-12 secreting cells may dilute out your signal. 

I can guess also, from where you isolate RNA and Proteines, if you're using in vivo model may be that cells from where you're isolate RNA were sitmulated by other cells secretion, may be that or not??? just a guess

Thanks a lot for answers.

Michelle, the cells i am doing experiments with are IC-21 mice macrophages.

It is well described that mRNA and cytokine levels may not be congruent. This is due to many of the causes given by the responding colleagues.

This discrepancy depends on several factors:

-The cell type used-the cells you are working with are no exception

-The stimulus applied including its t1/2, nature of binding to its receptor…etc

-Duration between applying the stimulus and collection of RNA and other samples

-Some stimuli activate RNA production transiently, with prolonged translation phase that results in ongoing cytokine production.

-The type of cytokine and its particular expression in the cell type studied

So I would not be worried about the fact that they are discrepant per se. One suggestion is to run a time course experiment and see if you can pick up timing of gene expression.

Another factor that needs to be clarified involve the technical details of the measurement of mRNA? I am assuming you are using qRT-PCR.

Have all experimental conditions been examined and optimized? Are all reagents in working order? Are you using a fresh master mix?....etc.

Hope this helps and good luck with your experiments

Thank you for your answer M.constantine Samaan.

Dear Omid,

According to my experience with other cytokines as IL28B, IFN-y, TNF-a, IL-10 and TGF-b, I agree with Korcan Ayata, Michelle Messmer and Brian Comer.