Hello everyone,
I have unexplained phenomenon in my data regarding one cytokine. The level of this cytokine by Qpcr is very high in knockout more than WT mice in naive condition and after inflammation. However, by flow cytometry, the level of the same cytokine at naive condition and after stimulation is less in KO mice than WT.
Can anyone help me to interpret this findings, with any references ?
Thanks a lot for your help
Hi,
remember that qPCR and flow cytometry assess completely different cellular endpoints. qRT-PCR measures mRNA, thus gene expression strength and immunological methods quantify protein amounts. Though both parameters usually are correlated with each other, there are limitations.
First, for a cell to accumulate a certain amount of protein from gene expression, time is required. Second, there are numerous post-transcriptional and translational regulation mechanisms in protein expression, which may lead to significant increase in your cytokines' transcripts, but not actually in increased protein amounts. For more complex regulation circuits, also a decrease on the protein level is plausible.
I would recommend to check the literature if there is anything known on the regulation of your cytokines' expression.
Hi
everything depends on your protein, experimental set up and conditions, it is impossible to answer to your question without detailed information.
As Patric Heinrich mentioned, changes in PCR are most pronounced after few hours and in protein levels in much longer time (usually days but depends on the protein).
But you mentioned that you are working with KO mice, maybe the KO caused lack of substance important for post-transcriptional regulation of your cytokine, so you have increase in mRNA, but no chance in protein levels (just hypothesis)...
I would recommend to rewiev as many studies about your KO target and cytokine pathways (both mRNA and protein levels) as possible to find out interpaly.
Hope this helps a little bit,
good luck
Thanks Matej Vicen & Patrick Heinrich ,
My KO mice is for knockout of a transcriptional factor which is mainly known as a negative regulator for many cytokines, but there is no literature that links this transcription factor and this cytokine together (we are the first to highlight the relation between them) , so it is difficult ti find literature specific for my study.
so I wonder if there is some literature for this phenomena ? or what should I do next ?
Thanks again.