According to the protocol, when designing the pre-miRNA double-stranded DNA oligo, the mature miRNA sequence used should be 21 nucleotides long (You may refer to Page 16 of the protocol here: http://tools.invitrogen.com/content/sfs/manuals/blockit_miRNAexpressionvector_man.pdf ). However, the mature sequences of miRNAs like miR-21, miR-200c etc often comprise 22 nucleotides or more. If I were to implement those 22-nucleotide sequences into the design of the ds DNA oligo, will it affect the miR expression later on?
I agree with Lye Meng Markillie!In my experience, for the expression of miRNAs, I have used lentiviral vectors by System Biosciences SBI (http://www.systembio.com/microrna-research/microrna-overexpression). I just cloned the genomic region of the miRNA of my interest (about 500bp). they work very well, resulting in a maturation of miRNAs in conditions similar to those of endogenous one.
Both vectors have been working in our hands for both microRNA-155 embedded shRNA mediated target-specific RNAi or expression of endogenous microRNAs. In that case it might be better to include the respective 5' and 3'microRNA flanking region as suggested by Felice to ensure maturation.
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