siRNA with low mRNA levels

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SUI
SUI's picture
siRNA with low mRNA levels

Hi everyone!

We do a lot of siRNA experiments with primary synovial fibroblasts and normally have no problems with transfection efficency (using nucleofection) or silencing (using 1-2ug of siRNA). Now, I tried to silence the chemokine receptor CXCR4 and it worked nicely in HeLa cells with 0.8 ug siRNA. The problem is that in synovial fibroblasts, even though they make functional protein, mRNA levels are around the detection limit (short half-life?). I tried up to 2 ug of siRNA and different time points, but I cannot see a change in the protein concentration. Does anyone know whether there is a connection between mRNA copy numbers/stability and siRNA efficiency? I asked Qiagen for advice and they told me, if there is enough mRNA to be translated siRNA should work. Well, it doesn't for me...

xangwang
xangwang's picture
Hi !

Hi !

How did you designed your siRNA ? is it a specific one or a bulk of 4 or more siRNA ?
to my point of view, raising the amount of si is not the only way for a good gene silencing.
of course it will depend on the size of the culture dishes experiment but for what I remember I used sirna in the ng range.

could you be more specific with your experiment ?

best

xang.