2 replies to this topic

[steffi333]

Hi again all,

I've been doing some experiments with my siRNAs, and have had good success in observing knock down by Western Blotiing after 24 hrs of transfection: my test siRNAs give good knockdown, while mock/untransfected and scrambled controls have no observable effect.

But now that I'm applying them in cell proliferation assays, I'm having problems. I can still get good knockdown with my test siRNAs, but the scrambled ones are also killing/inhibiting my cells! Even more than the test siRNAs in some cases I've blasted the scrambled sequences and can't see any reason why they would be having this effect.

Does anybody have any advice/experienced this before?

Can scrambled siRNAs have general non-specific toxic effect? I hope not, because I can't really afford to buy any more!

Some background:
-- I'm transfecting with Lipofectamine 2000.
-- I've done a range of siRNA concentrations (8, 16, 24 and 32nM), keeping the siRNA:lipofectamine ratio the same throughout. I'm not observing much cell death/inhibition in the mocks, so I don't think the amount of lipofectamine is the problem?
-- Cells are prepared by seeding in pen-strep free DMEM with 10%FCS, adhereing overnight, transfecting the next day, changing media after 6 hrs (to DMEM with 0.1% FCS and no pen-strep) and leaving for 7 days to proliferate before reading.

Thanks for any help anybody can give. I know I'm on this board a lot moaning about my silly siRNAs!  

Edited by steffi333, 04 May 2011 - 05:29 AM.

[pcrman]

This is not uncommon. Certain sequences are toxic to cells (or to just one type of cells but not another) for no reason. What you can do is to order several control sequences and use one that don't give you toxicity and affect the expression of your genes.

[steffi333]

pcrman, on 09 May 2011 - 07:31 PM, said:

This is not uncommon. Certain sequences are toxic to cells (or to just one type of cells but not another) for no reason. What you can do is to order several control sequences and use one that don't give you toxicity and affect the expression of your genes.

Thanks. I was worried that might be the case. I ordered two scrambled sequences and both are causing me problems, and my budget probably won't allow for me to purchase any more I shall have to have a chat with my supervisor.