Gene of interest targeting micrornas - (Jun/28/2013 )
I am trying to find some micrornas that target my gene of interest. I started by running the most common algorithms such as targetscan, pictar and microrna.org and i selected the best candidate micrornas. I then cloned them into vectors and transfected cells where i checked protein and mrna levels. However it seems none of this micrornas are downregulating my gene of interest.
Are there any strategies to screen (experimentally) the micrornas that target a specific gene?
Would it be possible do use a CLIP aproach overexpressing my interest 3UTR and search for enrichement of micrornas in the precipitated fraction?
Are the QI@GEN surefind plates a good alternative? They have HeLa cDNA of cells transfected with the complete mirnome mimics and i would check my gene of interest mRNA levels on this cDNA plates and search for downregulation. Do they work good?
My gene of interest has a huge 3UTR, more than 4kb, is it probable that no mir targets my gene?
Any suggestions for the identification of micrornas that target my gene of interest?
Thank you very much
It may be a better strategy to first transfect mimics for screen purpose and then validate by overexpressing precusor miRNA. That said, the Qiagen kit may be worth trying. In addition, mimics may generally give better knockdown efficiency on true target gene than vector. By the way, how did you clone your miRNA?
First of all, thank you very much for your reply and sorry for this very late response.
We could screen with mir mimics first, but individually ordering these molecules gets really expensive and does not guarantee results. With plasmids we can do it cheaper and quite easy. What i have been doing was cloning the pri-mir from gDNA and cloning it downstream of a H1 promoter. I validated the overexpression and i got like ~150 fold increase compared to a neg control. Still i didnt manage to reduce my gene of interest expression which actually has several binding sites... I am testing this with lipofection on HEK293T cells and evaluating the effects from 24 to 48 hours after, is this time enough?
Thank you very much
You'll need a postive control. Can you check the activity of a gene that is known to be a target of the miRNA you are expressing from the plasmid? If that known target is repressed but your putative target is not repressed, that suggests that your gene of interest is not regulated by the miRNA you are expressing. However, if neither the known target nor the putative target is repressed then that suggests something else is going wrong with the assay, for instance the RNA product of the plasmid might not be undergoing processing and loading onto RISC.
That is a great idea! And i actually have a qpcr primer assay for a gene that has been validated as a target of the same mir. I'll do a qPCR for that gene in the same rna samples.
Thank you for the suggestion!
Here, what did you validate: pre-miR or mature miR? If it is pre-miR, then you need to evaluate mature miR expression because some vector-expressed pre-miRs may not be processed into mature ones.
Thank you for your answer. I validated the overexpression using TaqMan probe assays which are specific for the mature microrna, so i think that is not the problem. Maybe i am not giving enough time for the "silencing" to occur? I actually couldnt yet check another gene as positive control because my primers are for mouse and i'm working on human cells now.
Any other ideas?