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? Could the QI@GEN surefind give some acurate results? I was thinking on testing maybe just 2 plates, one for my goi and other for a housekeeping gene. They apear to have the most common micrornas on the first panel. Do you think that just 2 plates would give accurate results? Thank you for your help
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!
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?
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?
I am starting to perform a dual luciferase assay in cells transfected with a plasmid containing my interest 3UTR bound to a firefly luciferase, and also a renilla luciferase gene with a plasmid overexpressing a microrna. I know that there are several commercially available kits for the determination of dual luciferase activities. However i would like to do the solutions of buffers, d-luciferin and coelenterazine by my own. does anyone have a protocol for the determination of firefly and renilla luciferases? I already have a protocol with buffers compositions for firefly luminescence detection, however i am not sure if this buffers will be compatible with coelenterazine and i don't want to risk buying it and than it not working.