I've got a bunch of miRNAs which predicted to be targeting some candidate target genes and I've been working on Luciferase assays to comfirm their real functional effect. I have selected some interesting miRNAs to proceed to the next step, which by blocking the binding site (seed sequence) of the miRNA to mRNA. Anyone has any experience how to make it (site mutagenesis) or where to buy it (target protector/blockmir)?
My another problem for site mutagenesis is to which nucleotide of the seed sequence should I mutate to? Also, what nucleotide should I replace so that the miRNA that I transfect wouldn't be able to bind to mutagenated site? Any software or 'rules of thumb' for this?
Any comments, suggestions would be very much appreciated. Many thanks!
Edited by altobarn, 14 July 2009 - 01:25 AM.