Hi there,

You can follow this literature.

https://www.sciencedirect.com/science/article/pii/S0167488917302574?via%3Dihub

In this I have used shRNA site which you can get using some literature study for your targeted gene and prepare the same.

Details have been covered in materials and methods. Good Luck.

Hi Roberto,

siRNA works well in fibroblasts when transfecting with lipofectamine. Depending on your readouts, however, just be aware that sometimes lipofectamine can lead to an inflammatory response in fibroblasts. I used to see spikes in TNFa following transfection. This can be largely avoided using nucleofection if inflammation could affect your read-outs.

Are the mRNAs you're looking to silence present in the cytoplasm or nucleus? If in the nucleus, siRNA won't work - you'll need to use LNAs.

since its primary cells, si-RNA would be the best with Lipofectamine. Depending of your results you can do double transfection as well.

Hi to everyboby!

Thank you for all you reply!

After your tips, I have asked to Thermo tech support and they suggested me the siRNA approach with RNAiMAX lipofectamine (I have seen some protocol for foreskin fibroblast).

I will try!

@ Mark Pearson:

My gene (EIF6) is a ribosome 60S bloking factor. It word as pre-60S maturation factor and it works molstly in the nucelus. I think that the mRNA should be in the cytoplasm and then the protein have to be imported in the nucelus to work. Bye!

All the best!

Roberto

I would propose to use lipofection via optimized systems (maybe optimized for small RNAs). Electroporation is mor efficient, however, many (sometimes most) cells will die during this process, which may be a problem in your exp. seting.

@ Andreas

Thank you!

The tech support of Thermo, suggested me the RNAiMAX lipofectamine and they provided me some articles in which somebody used this system with foreskin fibroblasts.

I have ordered the material. Hope all will be fine ;-)

Bye!

Yes, I think this is a reasonable suggestion/choice.