Problem with validation of the miRNA binding site in 3' UTR reporter - (Mar/25/2010 )
Hello Everybody
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?
Did you make 293T cells stably expressed your miRNA? If so, there is a possibility that the promoter used for miRNA expression was silenced.
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?
Hi,
I have a simlar problem like you. I overexpress both my tested miRNAs and reporter plasmid to screen for candidate that targeting my interested gene 3UTR. It works perfectly few month ago but when I try to repeat it now, it keep on failing. HELP!!!Everything is the same, medium, cells, plasmid, reagent. So, have you solve the problem with your experiment???
Thanks
luukasz4 on Thu Mar 25 16:35:32 2010 said:
Hello Everybody
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?
Hi.
I'm doing the same king of experiment but with other cell lines. Do you transiently transfect the 293t? how long after the transfection with the reporter plasmid you collect the cells for LUC assays? I noticed in my case that the extension of that time make some difference... for instance, at 24h I don't see a diference between the UTR and CTR construct, but I see it at early time points... ALso, the age of my cells made some diference! are your cells too long time in culture?
good luck