I have infected the mactrophage RAW 264.7 with E. coli and studied its NO and TNF alpha production in vitro several times which shows each of their low production respectively. However, when the mRNA expression is studied by semi quantitative RT PCR their band intensity is seen to be high saying that the expression level is higher whereas the final product formation is lower. Is this possible somehow? Can the infecting bacteria relate to the low product formation?
When are you measuring NO and TNF alpha? When I've used LPS to stimulate macrophages the TNF production is present at 4 hours and highest at 24 hours but NO measurement with Greiss reagent indicates peak at 48 hours. Besides the kinetics question there are definitely reasons why you might have mRNA but not product.
Hello Anne,
I had measured the activity of NO production for my mutants over 24 hrs and seen that it produces a peak at 8 hours which gradually reduces after that, so I consider that time for my experiments. Also, I have lysed the cells at that particular time only to isolate its mRNA, yet the mRNA produced is higher.
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