cellular siRNA localization - (Feb/25/2009 )
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!
I see a similar localization with labeled miRNA. But just to clarify, there isn't just one blog, but many, per cell. I'm not sure what compartment they are found in. First I thought about P bodies, but maybe it's too large for a p body? I guess it could also just be non-specific or aggregation of the RNAs?
The compartmentalization of transfection agent-siRNA in the "perinuclear region" is well known, typical of endosome-lysosome vesicles. The siRNA needs to get out of the compartment to be functional.
genehunter on Feb 25 2009, 10:09 AM said:
Presumably the fluorescent signal will be too weak and diffuse to observe once the RNA has left the vesicles?
Thanks for the feedback!
Yes, I guess it would then be very difficult to detect the fluorescence outside those compartments... Guess I have to wait and see if there's an effect kicking in