4 replies to this topic

[jhb80]

Hi all,

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.

[miRNA man]

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?

[genehunter]

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.

[miRNA man]

genehunter, on Feb 25 2009, 10:09 AM, said:

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.

Presumably the fluorescent signal will be too weak and diffuse to observe once the RNA has left the vesicles?

[jhb80]

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