just a very quick question from an RNAi newbi: just started to try and knock down my message of interest, so I was using fluorescently labeled siRNA oligos to test different transfection conditions on my HT1080 cells using Oligofectamine. I fixed the cells (PFA) the next day and had a look at them under the microscope: almost all of the cells (independent of the transfection conditions) had a massively bright signal (still detectable using an ND filter cutting out 90% of the light), however, in all cases this was a discrete 'blob' of fluorescence in the perinuclear region instead of a more homogenous cytoplasmic/nuclear distribution that I thought it should look like.
I was wondering if anyone could tell me if this is a normal localization pattern, or if there's a problem with agglomeration of my oligos? Also, should this be the expected localization, what's the cellular compartment that retains the oligos?
Thanks a lot!
Edited by jhb80, 25 February 2009 - 01:17 AM.