I'm dazzled over sth seemed to be able to do that less than one month but now it's more than three months and I cant reach to a conclusion. Any idea or tested protocol is greatly appreciated. I have some repeatable extremely significant data of Luciferase assay due to over expression of miRNA(Ambion pre-miR and 3UTR vector of its target).
But when I transfect cells(N2A) with different concentrations of miR,each time it produces a completely different result! two times a bit downregulating the target but the other two times up regulating the target a bit.
I have checked the expression of protein in the nontransfected cells and it is really well expressed. I even switched to using plasmids expressing pre-miR (having GFP reporter) but the number of GF^positive were too much low(transfecting premiRwith lipofectamin here is the details)
seeding 250000 cells per well 24hrs before transfection using 50,70,100umolar concent. of oligo
transfecting exactly as Invitrogen protocol scales adjusted for a 6well cell plate(cells are 50-60% confluent if not I wait until they reach to this confluency)
changing media between 12-24hrs after transfection.extraction of protein after 24 and 48hrs
(washing cells with cold PBS and all procedures on ice using ripa as chemical method of lysis)
plz share ur ideas and experiences while working with N2A mouse cells not only if in the context described above
Edited by gingerx, 20 August 2013 - 03:25 PM.