GFP siRNA transfection - readout - (Jan/24/2007 )
Hi everyone, I'm a newbie in siRNA transfection and hopefully someone can help me out.
In our lab, we would like to develop some new materials to deliver siRNA. In my experiment, I have 2 eGFP expressing cells which I would like to use siRNA to silence the GFP. I'm trying to use the GFP fluorescence (480/520) as a readout parameter to determine the efficiency of transfection. I'm working on a serum free transfection in a 96 well plate, where I put the siRNA complexes in HEPES with DMEM for transfection. After 6 hrs, I replaced the transfection medium with normal growth medium (DMEM, 10% FBS, no G418) I used the plate reader to take the GFP fluorescence after 1, 2, and 3 days. For some reason, the wells with just the medium contributed a lot of fluorescence, so the sensitivity is very low and not quite useable.
is the high baseline value contributed by the phenol red in DMEM? in that case, should I replace medium with PBS before I read the plate? or should I get some DMEM with no phenol red for this experiment?
do I have to lyse the cells before taking fluorescence reading?
is this method applicable, and give enough sensitivity? or should I monitor mRNAs for GFP instead?
Thanks for your input in advance.
usually you could also Facs your samples but it seems to be a more High-throughput assay you perform.
However if you really want to show transfection efficiency I would use an alexa-fluor labeled siRNA which can also be read around 480nm because the silencing itself is not really accurate by measuring the GFP.
You can basically show that the silencing itself is working with the GFP measurement but I would rather go for a conjugated siRNA to show the performance of your transfection method.
And yes, it's the phenol red. DMEM without phenol red is better, PBS is best. In my experience, anyway.
U could try to replace the DMEM with PBS before reading the values and replace the media later.
Have you thought about trypsinising cells and doing FACS to identify the GFP positive cells. This would give you actual cell number.
I am not sure if this is feasible for your experiments.
It seems luciferase might provide better signal-to-noise than GFP; this would eliminate any problems with fluorescence from medium components or autofluorescence. Ryzard Kole's antisense upregulation assay systems often use a luciferase reporter, and we have used these systems to optimize delivery of Morpholino antisense oligos. Using his splice-correction strategy we can see an increase in luciferase signal as the endpoint of antisense activity, which makes it easy to distinguish antisense activity from toxicity; unfortunately that would be a difficult system to engineer with siRNA, but at least you could take advantage of luciferase readout to get around your current fluorescence background problems.
Some references to Kole's work:
Here is a recent example of the use of Kole's splicing upregulation lucoferase reporter system for oligo delivery assessment:
Thx everyone for the input. After my best attempt to refine my technique, I still don't see any significant difference in fluorescence from the plate reader, even though the silencing can be clearly seen with a Flu microscope. So, I've gave up on the plate reader and will be switching to flow soon.