Hi all,
recently I made a weird observation in FACS and it would be great to have some opinions on that.
I transfected some small RNAs into cells and measured the cell cycle distribution using PI staining.
Staining looks fine; however in one situation (out of 4 small RNAs) I see a shift of the complete cell cycle staining on the x-axis; the distribution in the single phases look 'exactly' the same compared to the control but the complete graph is significantly shifted.
I assume that the transfection of this specific small RNA causes the cells to become aneuploid. The effect seems to be very specific to this one small RNA.
Can this be an artefact, or do I miss something about the PI staining? How to further prove this idea?
Thanks for any suggestion,
Chhers
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Posted 30 July 2009 - 05:07 AM
Bomber, on Jul 29 2009, 06:41 AM, said:
Hi all,
recently I made a weird observation in FACS and it would be great to have some opinions on that.
I transfected some small RNAs into cells and measured the cell cycle distribution using PI staining.
Staining looks fine; however in one situation (out of 4 small RNAs) I see a shift of the complete cell cycle staining on the x-axis; the distribution in the single phases look 'exactly' the same compared to the control but the complete graph is significantly shifted.
I assume that the transfection of this specific small RNA causes the cells to become aneuploid. The effect seems to be very specific to this one small RNA.
Can this be an artefact, or do I miss something about the PI staining? How to further prove this idea?
Thanks for any suggestion,
Chhers
recently I made a weird observation in FACS and it would be great to have some opinions on that.
I transfected some small RNAs into cells and measured the cell cycle distribution using PI staining.
Staining looks fine; however in one situation (out of 4 small RNAs) I see a shift of the complete cell cycle staining on the x-axis; the distribution in the single phases look 'exactly' the same compared to the control but the complete graph is significantly shifted.
I assume that the transfection of this specific small RNA causes the cells to become aneuploid. The effect seems to be very specific to this one small RNA.
Can this be an artefact, or do I miss something about the PI staining? How to further prove this idea?
Thanks for any suggestion,
Chhers
Hm, what about transfecting cells with this RNA, staining the DNA and looking at them microscopically? I've never done this but it would sound reasonable to me.
Is the "new" G1 on the same position where the G2 of the other cells appear or trice G1 etc.? If not, it doesn't seem to be polyploidy. PI staining intensity sometimes shifts even upon treatment with normal drugs without any clear reason. I had to adjust the settings for treated cells as they always shifted a bit to the right and in this way, it was impossible to do an overlay with the untreated ones. Maybe the small RNA changes the DNA in a way that the DNA is now able to retain more of the dye? Just as an idea.
