Lipofectimine? How to optomize?Some basic Questions? - (Jan/15/2006 )

Hi All,

I am doing siRNA knockdown for the first time tomorrow. I have pre-designed siRNA to my gene of interest from DHARMACON. I got recommendations from many to use LIPOFECTAMINE 2000 transfecting reagent from Invitrogen rather than the ones provided by DHARMACON.

1- How do I modify the protocol given by Invitrogen for their "stealth siRNA" for the ones provided by Dharmacon? Can I just simply use "exactly" their protocol for lipofectamine and replace their stealth siRNa with my Dharmacon siRNA?

2- I have been advised by many to do siRNA transfection when the cells are in “susupension”. As in, split the cells the same day, plate them in suspension at the appropriate cell density, and the same time do the siRNA transfection. But all the different protocols given by invitogen say that the cells should be plated “one day before”. Has anyone done siRNA transfection in suspension?

3- I am not using any of the cell line given on the Invitrogen website !! I am using a primary fibroblast culture. Does it matter if it a cell line or primary culture? Which of invitrogen’s protocol do I use? How do I modify invitrogen’s protocols to suit the primary cell culture I am using?

4- How do I know what cell density to begin with?

Many thanks in advance!!

RNA

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I have done hundreds, if not thousands, transfections mostly using lipofectamine 2000 and siRNA synthesized by Invitrogen (not stealth siRNA).

According to Invitrogen, Stealth siRNA is siRNA with chemical modification to minimizie interferon response and increase its stability. Although I don't know what exactly the modification is, it makes the siRNA no much difference from other siRNAs. So the protocol for stealth siRNA should work for siRNAs synthesized by others.


Yes you can do transfection immediately following plating cells and before they have settled. But I prefer doing the transfection the next day because the cells are already stressed during the splitting and more cells will die if lipofectamine is added at this time. Therefore I would suggest that you follow whatever the protocol says.

Primary cells are known to be refractory to transfection and I don't have much experience with this. Some companies boast transfection reagent capable of transfecting primary cells. You may look around.


Depending on different cells. The rule of thumb is to plate cells at a density so that at the end of transfection, the cells should be 100% or less confluent.

Hope that helps.

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pcrman....this question is for you.

How much of lipofectamine do you use in a 24 well experiment with siRNA?? is it accoridng to ug/well (as in the case of plasmid transfection) or in terms of uM siRNA??

thnx

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Hi Pria,

I usually use 5~6 ul/well Lipofectamine 2000 for an siRNA concentration of 1~50 nM in a 24-well plate as suggested by the vendor. Although this amount is good for a wide range of siRNA concentrations, if more siRNA is transfected, the amount should be increased accordingly.

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thanks...

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