I have cloned a 5 Kb 3'UTR of my GOI into a single XbaI site of a pRL-CMV vector. I want to see binding of microRNA with that but I got my insert in an opposite direction (luc-3' end of UTR--- 5' end of UTR). Does it have to be in the same orientation as natural mRNA for microRNA targeting?
agree with Rikard and ricardo, I can add that you could use the construct as a negative control..
I agree, must be in the same orientation: I typically clone the sequence in opposite direction as negative control.
cheers D.
It must have the same orientation. If it were in the reverse orientation, then the specific microRNA, whether it has a 5p or 3p designation, will not bind to the messenger RNA because a 5p or 3p microRNA is single-stranded.
It must be in the same orientation. MicroRNAs bind to single stranded mRNAs. A different cloning orientation means a different mRNA sequence.
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