miRNA luciferase reporter assay queries - (Jul/23/2007 )
I am a first year PhD student and am new to the filed of miRNA. I want to overexpress miRNA and see the phenotype of the cell and also want to knock down miRNA and see what their effect is with the use of luciferase reporter assay. I was going through varoius companies and found that Ambion has 'Anti-miR™ miRNA Inhibitors ' and 'Pre-miR™ miRNA Precursor Molecules' for inhibition and over expression. Also, they have 'pMIR-REPORT™ miRNA Expression Reporter Vector'. I don't want to use shRNA. Has anybody used any of these products? Also, what othe alternatives i have for my experiments. Last, where can I get a detailed protocol for the above experiments?
Thanks in advance.
I've used the Pre-miR molecules and the negative control #1 molecule. Pretty easy to use, just transfect like a normal siRNA, I used lipofectamine 2000 and used the standard protocol included in the manual (although I checked transfection efficiency by knocking down a GFP reporter with siRNA against GFP in a pilot experiment first). Dharmacon has an equivalent product, not used them or seen any publications with them just yet.