I did overexpression (Roche Xtremegene) and knockdown (shRNA) of one transcription factor in Hela cells and harvest cells for RT-PCR after 48h.
Now we can see that knockdown of TF can induce IL-10 mRNA increase (40 fold) and iNOS mRNA decrease (15 fold). But both genes have no change in overexpression comparing to empty vector control.
We noticed Xtremegene reagent itself (all transfection group using it show same increase) can induce huge change in mRNA level (2000 fold) comparing to NT sample but shRNA has no any impact.
Anybody can give some suggestions?
Overexpression of a transcription factor does not always correlate with the overactivation of its target genes. It can have different reasons for this phenomenon: lack of important cofactors, need for activation of transcription factor (for example through posttranslational modifications) or simple saturation of all potential interaction partners (if the overexpression is too strong).
Thank you Eugen.
You mean the results of knock down or knock out are more informative than over expression? Can I said the TF I knocked down definitely be involved in the regulation of IL-10 since its mRNA changed over 40 fold?
Beside what Eugen said, have you ever considered the non-specific/off-target effect of your shRNA? Did you try multiple shRNAs and get similar results?
Thanks Xiaojiao
Yes. We used two shRNA from different companies and got the same results.
I noticed all the inflammation related genes including IL-10, iNOS, IL-1b and TNF alpha increase so much after transfection with X-tremegene HP, even in the empty vector control (unfortunately I didn't set a control of transfection reagent alone). And other genes such as Bcl-2, TRAIL didn't. (from the same experiment and samples).
Does anybody know transfection reagent has such a huge impact on inflammation related genes?
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