I could visualize cell death after 24 hours in the siRNA treated wells while the transfection control and untreated cells were looking fine. RNA yield was also better in the controls. What could be the probable reasons for such a low RNA yield from siRNA transfected cells.
It depends upon how much concentration of siRNA you are using for the treatment before performing your main experiment you have to do the MTT assay and find out the appropiate concentration of siRNA should be use.
Since your controls are working fine the error is most probably with the concentration of siRNA ,you have to optimize the siRNA conc. first.The reason behind low RNA yield would be the same
Another possiblity that might be your siRNA is against some housekeeping genes ,silencing of which cause lethal mutation leads to death of cells.
Hope this information may help you
Hi Lakshmi,
First I did not get what you mentioned as transfection control. Is that a non-targeted control or a control that will kill most of the cells (positive transfection control)?
Generally, I use both in addition to my target siRNAs. I used to see ~90% death in my positive transfection control within 24-48 hrs indicating the experiment is fine.
A word of caution is that most of the siRNA have off-target effect, so I used to titrate and check which one used to work in lowest concentration. Please let me know where are these from, I have experience with qiagen and thermo.
Best,
Abhilash
Hi Abhilash,
My transfection control is the cells where I just add the transfection agent without any siRNA. The siRNA I am using is from Ambion. I had chosen this siRNA as it had a back reference.
Hi Lakshmi,
What is the final concentration? What is the Ambion suggested concentration?
Abhilash
Hello Lakshmi,
Which gene are you targeting? I did a RNAi screen to cause apotosis in cancer cells and this sounds like your cells go apoptotic shredding the RNA into single bases who cannot be extracted and this results in lower RNA yields as compared to the references because this cells are fine. Thats a logic consequence of apoptosis.
You can also estimat how many cells are dead after your incubation time by comparing the RNA yields, maybe untreated cells have delivered 4 µg total RNA in sum and treated only 2 µg then you can estimate that 50% of your cells die due to siRNA silencing.
Another thing is that the transfection reagent is not toxic when you do not add a scarmbled siRNA control allowing the formation of lipoplexes delivering the RNA into cells distrubing their metabolism and viability. I used a final concentration of 2.5 nM scrambled siRNA control in 200 µl final volume in a 96 well plate with 0.15 µl Lipofectamine RNAiMAX and this was well tolerated when I seeded 3-5000 cells/well.
I hope this helps.
Regards Volker
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