Different siRNA transfection agents - Have enyone tested them??? (Dec/16/2004 )

is it possible to use PEI?

Seb_

I have worked using siRNA with Jurkat cells and K562 cells in my previous lab. For such suspension cells, normally lipid-based transfection reagents have presented very low transfection effiencies. I have tested OpenBiosystem Arrest-In and Roche x-tremeQ2 (for K562, Jurkat and HeLa), both got <10% transfection efficiency, and Arrest-In is more toxic.
For that reason, we turned to eletroporation-Amaxa Nucleofector, after optimization, for early passage Jurkat cells (<10), we could get >90% efficiency.
Good Luck!
kevin

Most publications are using Lipofectamine 2000 and Oligofectamine (Invitrogen) for RNAi studies.
You might need to optimize it a little bit.

If you are studying RNAi oligos, the following link have different transfection protocol for different cell types.
http://www.invitrogen.com/content.cfm?pageid=10084

Hope you find it useful.

I've been using calcium phosphate for BAEC, it's working great!! I guess that's the cheapest you can get.
In my lab, we're basically using calcium phosphate for most of cells (HeLa, HEK 293, BAEC, NIH 3T3...). Post if you need any protocol.

hi, does any one has idea how long could be attR1 and attR2 site which are used for LR reactions. If you ever have sequenced it than please can you tell me exact length of it??
Many thanks.
Neesheel

The experience from our lab is that the TransIT-TKO from Mirus (Dhamarcon) works better than other transfection reagents tested by us so far, such as Lipofectamine 2000, oligofectamine (Invitrogen), Fugene 6 (Roche), RNAiFect (Qiagen) and siLentFect (Bio-Rad). Since our experiment was performed on a few of cell lines including NIH3T3, MEF, Hela, 293T, ST14A etc, other cell-dependency with respect to different transfection reagents should not be excluded. But by transfection of fluorescent tagged siRNA, under microscope, the best result generally came from TransIT-TKO.

We would like to share our experiment with peers in the same field, good luck.

I have a similar problem, we try to transfect - only for testing transfection - right now with luciferase expressing plasmide (pGL2, pGL3) in monocytes (human, primary cells derived from periphery blood). Good thing about luciferase is, it has a short half life so we can estimate the time the plasmide is really actively inside the cell.
With Gfp it works without any problem but Gfp is of course stable like hell and accumulates so no real knowledge gained here.
What we tried for three times now is amaxa nucleofection human monocyte kit. It is expensive - now even more than before (330 euro ...) - and not any luciferase activity could be measured. Not at 12h, not at 24h, not at 40h, not at 48h and not at 72h.
Both plasmides work well in cell lines when electroporated.
We are looking for alternatives now, cheaper ones preferably.
Anyone has good results so far with primary monocyte transfection and maybe even luciferase?
Thanks for your help

I would like to add, that Lipofectamine2000 by Invitrogen is a godd, but very stressing reagent to some cells. Metafectene (Biontex) is not as toxic. Lipofectamine works good for cell lines which could not be transfected with other reagents..

Hi everybody,

I also have problems with transfecting my melanoma cells. I have tried Ambion, Qiagen, peqlab and Novagen. Nothing worked very well so far. I would like to try calcium phosphate. Does anybody have a protocol which he/she would be willing to share with me?

Thanks so much.

Hi all....

I used to infect jurkat cells with Rnai-vector (using calcium phosphate) but everytime I did it the jurkat T cells always seems not happy.... I suspect that the reagent is toxic to the jurkat T cells...

Is anyone know what is the best reagent for jurkat cells?

thx a lot