The vector I'm using is pmirGLO (7350pb) and my 3'UTR region has 2300pb (yes, it is very long). I am testing the interaction of 2 miRNAs, both have two binding sites to the 3'UTR region, two of them very close and the other two very far between them.
I cloned all 3'UTR region and perform a luciferase assay comparing against the empty vector. However, the empty vector throws on RLU 100.0000, while the vector with the 3UTR only RLU of 10,000 (both luciferase and renilla), I do not know if this magnitude is very low. I suspect that the efficiency of the trasnfection is low by the size of the vector with the 3'UTR. I was thinking cloning only the target region of my miRNA, but I think that the presence of the two regions may be necessary for regulation to be efficient.
On the other hand, I am building the mutated controls, and I have the doubt whether to work on the whole 3'UTR region or just on the target regions. For this, I want to buy the QuikChange Lightning kit.
Can someone give me opinions? thank you very much!
Dear Kathleen
You are probably facing a problem of efficiency in the transfection as you suspected. For such a long UTR I would advice you to chop it out into pieces including only your target sites. It is somehow non-physiological, but you should get better transfection efficiency.
Luciferase assays are very artificial, and I am not a particular fan of them. Remember that you will change the physiological environment of your UTR probably using a different cell line, and also removing the rest of the mRNA.
I would rather prefer to use AgoIP analysis to test for enrichment of your targets.
Best
Thanks for your answer Francisco. Indeed, in addition to the luciferase assay I would like to perform an AgoIP analysis.
I will take your recommendations.
Regards
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