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DNA and siRNA transfection and cytotoxicity

transfection

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#1 Guest_Grigo123_*

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Posted 22 October 2012 - 10:44 AM

Hi All,

I am designing a big study in A549 cells and have some problems with picking up an appriopriate transfection agent. I need something that:

1) Works for both siRNA and antisense DNA since I will be compering the results.
2) Has the minimal possible toxicity and effect on gene expression - to get this I am willing to work to accept long and complicated protocol.
3) High knockdown (>=90%) in plate format (adherent cells).

So far I have used Lipofectamine 2000 but after some reading it seems very toxic and induce a lot of genes. Any idea what would be the best (liposome, non-liposome, calcium phosphate)? Most of online comparisons are made by the companeis themselves so can't really trust those results too much.

Also does anyone know if factors in medium such as high glucose or sodium pyruvate affest the transfection? I was thinking about using serum free media insetad of optiMEM to reduce toxicity but still need the 90% knockdown.

Thanks,
Grigorij

#2 bob1

bob1

    Thelymitra pulchella

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Excellent

Posted 22 October 2012 - 12:18 PM

I have found RNAiMax from Invitrogen to be quite good, much better and far less cytotoxic than Lipofectamine 2000. The knockdown is dependent on the siRNA that you are using, not the transfection reagent.

You don't need the OptiMEM in my experience, I typically just use the medium I grow the cells in just use the same medium without added serum.





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