I transfected the clathrin siRNA at 20nM and 50nM for 2 to 6 days, then lysed the cells, then did the western blot. The image is in the attached annex. The best kncokdown efficiency I can reached is 70%, but how can I make the efficiency better? Thanks very much!
You may need to think about your transfection reagent used or the timeline- siRNA is transient and if you have a dividing cell line, you will lose the siRNA quickly. So you may think about your incubation time with your cells. You may need to try to lyse the cells directly after siRNA removal or during the incubation period- say 2 days.
if all didn't work out, you may need to look at your oligo to think about enhancing stability of your siRNA; you may, in the process, get some increase in efficiency. Good luck
You might use instead several SiRNAs instead of only one. Using a combination of SiRNA for the same target gene might have a positive effect on your knockdown.
Good luck.
Best,
Hamadi
Thanks very much Hareth Wassiti! For the timeline, the cell line Hep3B replicate in each 48h, and I lysed the cell everyday from 2 to 6 days after the transfection. If I understand well, your proposition is to lyse the cell also 1 day after the transfection?
Thank you, Hamadi Madhi! The provider just gave us only one siRNA, was it normal? So in your opinion, perhaps we should buy some more ?
yes , of course Liu and you d better design it by yourself. I strongly recommend using SiRNAimax Lipofectamine reagent.
Just perfect!
Okey, I will disccus this with my director and try to design it by ourselves. We did use the Lipofectamine reagent. Thanks!
Liu. Generally, most of your siRNA will be expelled out of the cell via exocytosis. This may not be a huge issue when you have low expression of the target. but if you do have a high expression, it may be problematic. but looking at your bar-graph, I would think the time is not an issue. It's more plausable to vary the concentration of your transfection reagent or increase the siRNA at 50nM and measure 2-4 days post transfection. Transfection reagents are the devil! they vary dramatically between cell types. contact your transfection vendor and ask them specifcally regarding your cell-line.
Best of luck
Harry
OzBiosciences.com have a reagent finder according to the cell types that you ant to transfect and the vectors that you want to use. Lullaby seems appropriate for you. Ask for a free sample on their website.
http://www.ozbiosciences.com/transfection-sirna/24-lullaby-sirna-transfection-reagent.html
Good luck!
Romuald
use the best reagents and optimize the transfection protocols. Silencer Select siRNA (potent and specific) + RNAiMax reagent
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