No, in this case i did not transfected cells with miRNA or miRNA constructs. I just transfected 293T cells with my 3'UTR luciferase construct and it was repressed through endogenous miRNAs. It seems therefore, that in my recent experimetns additional repressors occurs. But how, when previously mutation of the sites for this only one miRNA in my 3'UTR construct cause clear de repression?
I have one problem with my luciferase assay concerning validation of the miRNA binding sites in the 3' UTR of the gene. I hypothesised, that one miRNA type repress translation of my gene through binding to its 3' UTR. Therefore, i made a construct with this gene 3' UTR cloned after the Firefly luciferase gene. After transfection in 293T cells I found indeed repression, when compared with positive control (construct without 3' UTR sequence). Then i have mutated all 4 known (according to TargetScan) sites for this miRNA in the construct. Luciferase assay indeed indicate significant de-repression (around 30-40 %) and this result was repeated several times. However, since few week i do not see any derepression for this mutated construct anymore! All conditions, constructs, reagents are the same. Could it be that that the cells have changed themselves so much. Or, what could be the reason? Do You have similar experience?