About the conc. use of the siRNA - (Oct/13/2006 )

Dear all,

I'm a beginner in using siRNA. I'm so confused about the concentration I need to use in each assay. Would anyone can tell me the detail protocol of it? I'm using a 100uM siRNA stock and lipofetamin 2000 to silencing a gene in HepG2 cells.

Thank you so much!

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i think you should find everything you need on the product information sheet that came with the lipofectamine or on the website of the company where you bought your siRNA.

there is not correct concentration for all cell lines so you will have to use varying concentrations to start off with until you decide upon the one which gives you the best protein knockdown and cell viability, i would start between 20-50 nM (final concentration) and play around until you get satisfactory results..


hope this helps

enjoy

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Thank U so much!

I've tried at 50nM last week, but it leads to 40% death in the transfected cells. I'll try at 20nM again. Thank U so much!!

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For RNAi, usually a siRNA concentration ranging from 10-100 nM can be used. The concentration is the molar value of siRNA per litre in the final transfection medium. For example, for each well of a 6-well plate, if final transfection volume is 2.5 ml, 6.25 ul of siRNA (20 uM stock) will be needed to achieve a 50 nM transfection concentration.
If you see large scale cell death, you can also decrease the amount of lipofectamine used.

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Thank U for your suggestions so much !!

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