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Weird effect of siRNA on protein in cells


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#1 runnerman

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Posted 01 October 2010 - 08:57 AM

Hi all,

I am experiencing a weird problem with siRNA knockdowns in primary human T cells.
I electroporate the cells with a BTX square wave electroporator - transfected with control siRNA or siRNA to target of interest (Dharmacon siGenome siRNAs).

After 48-120 hours I monitor knockdown of my target protein by Western blot.
The weird thing is......there is always much less protein in my target protein lane (>80%) as hoped, however, I usually see a similar reduction in expression of other proteins in this lane e.g loading control such as Actin or Tubulin. The control siRNA (siGenome) has no effect on my protein of interest or on the corresponding loading control. So, this effect is not due to electroporation. It seems that the siRNA to my target of interest is shutting down global protein translation, an effect that would not be predicted from this protein, and the cells are still viable! I am using a high conc of siRNA however for both control and target siRNA - 1uM. Has anyone ever observed something like this?

Cheers

#2 Dukey

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Posted 01 October 2010 - 04:15 PM

Hi all,

I am experiencing a weird problem with siRNA knockdowns in primary human T cells.
I electroporate the cells with a BTX square wave electroporator - transfected with control siRNA or siRNA to target of interest (Dharmacon siGenome siRNAs).

After 48-120 hours I monitor knockdown of my target protein by Western blot.
The weird thing is......there is always much less protein in my target protein lane (>80%) as hoped, however, I usually see a similar reduction in expression of other proteins in this lane e.g loading control such as Actin or Tubulin. The control siRNA (siGenome) has no effect on my protein of interest or on the corresponding loading control. So, this effect is not due to electroporation. It seems that the siRNA to my target of interest is shutting down global protein translation, an effect that would not be predicted from this protein, and the cells are still viable! I am using a high conc of siRNA however for both control and target siRNA - 1uM. Has anyone ever observed something like this?

Cheers


1 uM is wayyyyyyyyyy too high for siRNA. 100 nM is the upper limit of what is acceptable to be honest. I am not surprised by your problems with that concentration of siRNA. Why oh why are you using 1 uM?

#3 pcrman

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Posted 01 October 2010 - 04:22 PM

Even synthetic short siRNA has been shown to induce interferon response by activating PKR which is a global repressor of protein translation.

#4 runnerman

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Posted 04 October 2010 - 12:34 AM

Thanks for the reply. The conc of siRNA used in elctroporation is usually much higher than standard lipid-based transfection, due to the inefficiency of the electroporation technique. For example, Amaxa say to use up to 300nM siRNA for electroporation of siRNAs into T cells, and I've read many papers that have used up to 1uM siRNA to electroporate T cells. A dose-response of siRNA is the way to go I suppose.

Cheers




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