Hi,
I am doing FACS experements to assess the GFP inhibition by siRNA. I am using A549 cells that are producing GFP and treated the cells with siRNA to inibit the GFP production. the cells then analysed by FACS.
How can I report the percentage of GFP inhibition? is it just as a percentage of median FITC of treated cells to the untreated cells?
thanks
As you have correctly said one way would be to take the median GFP fluorescence of the entire population which indeed should be lower in the silenced/treated samples. The other way to do it would be to gate the GFP positive population and look for a drop in the % of that population in the silenced/treated samples.
It's always more convincing to also include an analysis where you've mixed control and silenced cells together and see a bi-modal distribution. To calculate the true percentage, presumably you will need a negative control (non-GFP expressing cells: 0%) and a positive control (GFP expressing cells: 100%). If you're not sure about the linearity of your measurements, you could probably mix together these two populations in various ratios to establish a curve and see where your siRNA knockdown cells lie on this standard.
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