I am currently having problems performing my cell functional assays since when I reverse transfect my cells (HUVECs) my knockdown displays high cell death. In contrast when I transfect them by the standard forward transfection I dont see any significant cell death in my knockdowns (same concentration of siRNA used in both and same cell seeding ratios). This eventually gives out differences in thereadout of my functional assays such as proliferation. I am unable to figure out such huge discrepancy and eventually decide which method to go for? Since with reverse transfection I get some functionality of my knockdown but contrary during forward transfection I see no significant differences. Any inputs on it would be helpful.
Thanks
Hello Akshay,
I routinely use reverse-transfection for transient silencing with siRNA. The cells I work with (differentiated adipocytes and myuotubes) are quite sensitive to the detachment process, but when I evaluate the cytotoxicity , it is always below 10%. Some suggestions :
- the siRNA itself is hardly toxic. When I optimized my assays, I used 250nM siRNA for forward transfection and 25nM for reverse, using the same concentration of transfection reagent. However, I got only 50% reduction with forward and 90% in reverse. So I'd suggest - keep the siRNA concentration the same, but reduce the transfection reagent concentration as it is much more toxic.
- another possibility is that you over trypsinize the cells. For trypsinization I always wash 2 times with plenty of PBS to remove traces of serum and then use the minimum concentration of trypsin possible.
- you could replace trypsin for Accutase. This is a wonderful product I recently found and started using. Unlike trypsin, you don't risk to damage the cells, as this product degrades only the extracellular matrix, but not the surface receptors. In addition, you get a wonderful single-cell suspension and when you reverse-transfect it, the silencing is even better!
Hope that helps.
Hello Akshay,
I routinely use reverse-transfection for transient silencing with siRNA. The cells I work with (differentiated adipocytes and myuotubes) are quite sensitive to the detachment process, but when I evaluate the cytotoxicity , it is always below 10%. Some suggestions :
- the siRNA itself is hardly toxic. When I optimized my assays, I used 250nM siRNA for forward transfection and 25nM for reverse, using the same concentration of transfection reagent. However, I got only 50% reduction with forward and 90% in reverse. So I'd suggest - keep the siRNA concentration the same, but reduce the transfection reagent concentration as it is much more toxic.
- another possibility is that you over trypsinize the cells. For trypsinization I always wash 2 times with plenty of PBS to remove traces of serum and then use the minimum concentration of trypsin possible.
- you could replace trypsin for Accutase. This is a wonderful product I recently found and started using. Unlike trypsin, you don't risk to damage the cells, as this product degrades only the extracellular matrix, but not the surface receptors. In addition, you get a wonderful single-cell suspension and when you reverse-transfect it, the silencing is even better!
Hope that helps.
But just one remark if the concentration of the transfection reagent (lipofectamine 2000) i use was the reason for cytotoxicity then the scramble control cells should also display such high cell death. I seem to observe this phenomena just in my knockdowns that when I reverse transfect the cells under go massive cell death but with the forward transfection they are quite well.
But as suggested I would definitely try using accutase and reduce the transfections reagent.
Thanks
What was the silencing in % in your forward and reverse transfection? Maybe the gene you're trying to silence is essential for the cell's function and getting "too much" silencing in your reverse tarnsfection results in cell death only in the siTarget, but not in siScramble - treated cells?
in forward transfection i havent assessd the % yet but in reverse it was about 95%
Well petar thanks for the tips they really helped. Specially the use of accutase along with reduction of transfection reagent works well now. The forward transfection efficiency was indeeed really low.
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