Are miRNAs phosphorylated at the 3' end? - Labelling of miRNAs in a T4 RNA ligase reaction (Nov/17/2006 )
I would like to know if mature miRNAs are phosphorylated at the 3' end or not? And this leads to one more question: Is it needed to dephosphorylate (by alkaline phosphatase treatment) the RNA before labeling with a linker in a T4 RNA ligase reaction?
miRNAs are actually phosphorylated at 5' end
In ligation using pre-actived linker (available from IDT Inc), dephosphorylation is not required, otherwise, dephosphorylation is needed for avoiding ligation of miRNA to other RNA fragment and selfligation.
Following link is an example of labeling of miRNA by ligation based technqiue:
Dear Rye: Thanks!
The paper you have kindly linked to was the very reason for my question.
I'm not entirely sure what you mean by pre-activated linker?
In the cited paper they link pCp-Cy5 or pCp-Cy3 to the 3' OH of miRNA (and all other RNAs with free 3' OH).
We are labeling miRNA by a slightly diferent linker, however, in principle it's the same. We do not dephosphorylate before ligation (but maybe we should!). I beleive that the excess of linker to endogenous 5'P RNA ends is very high and that self ligation will not be an issue. But maybe we should actually test it!
pre-activated linker oligo with 5' adenylated can ligated to miRNA in a reaction condition without ATP, so avoid circularization of miRNA and non-specific ligation
while synthesis of pre-activated linker is costly...