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RNAi in CHO cells - (Jun/28/2005 )

Hi ,
I want to ask you if we tried RNAi in CHO cells. I am using in vitro transcription to produce ds RNA, then I cut the ds RNA with Dicer enzyme. Transfection with Lipofectamine 2000 with fluorescent oligos gives me a high percent of transfection 90%. When I transfected CHO cells with specific short ds RNA the 50 % of cells died, after 72 hrs. They start dying after 24 hrs.
Is this interferon response?, because the Dicer si 100% efective, and I not really sure that I serapate the long ds RNA from sh ds RNA. I tried different columns, but still not sure.
I also tested the Lipofectamine 2000 toxicity, I know this is toxic for the CHO cells, I figure out that 4 ul/180mm coverslip is fine.

I going to run the shdsRNA on polyacryamidic gel, and extract shRNA from the gel. But still I don't know if I can obtain RNAi effect on CHOcells.
thaks a lot

-sabcs-

Hi,

I've never transfected CHO cells with siRNA. How is your mock transfection doing on CHO cells. Do you include any control siRNA? Only after you have included these two, will you know if any effect is sequence-specific. Why don't you just use synthetic siRNAs which is pretty cheap now and can save you lot of pain and time.

-pcrman-

QUOTE (pcrman @ Jun 28 2005, 08:40 PM)
Hi,

I've never transfected CHO cells with siRNA. How is your mock transfection doing on CHO cells. Do you include any control siRNA? Only after you have included these two, will you know if any effect is sequence-specific.  Why don't you just use synthetic siRNAs which is pretty cheap now and can save you lot of pain and time.


Hi and thanks for reply.
I am working now on control for siRNA. The problem is for CHO cells we don't have the genomic seq. In am trying to design specific primers for one gene ( I compare the seq from mouse, human, and try to find the conservative region of the gene), add T7 promoter to the primers, PCR with genomic DNA, now I started with cDNA, and do the transcription in vitro.

I am looking for nuclear proteins, but my big concern is that the CHO cells are hard to transfect with shRNA and that I can not find the right seq of the gene for RNAi.
thanks a lot again

-sabcs-

Hi there!

I have a question... you said that you transfected CHO cells with Lipofectamine and got 90% efficiency...Which protocol are you using? (Medium at transfection, amount Lipo, amount DNA,...) I'm working with CHO cells as well and only get 20% efficiency of transfection...

Thanks Stardust

-stardust-

Same here. I only got ~30% efficiency with my 3 siRNAs using lipo2000 on CHO???
Almost give up lipo2000 now.
I'd appreciate if you can specify your protocol.

-fair view-

QUOTE (fair view @ Nov 3 2005, 02:18 PM)
Same here. I only got ~30% efficiency with my 3 siRNAs using lipo2000 on CHO???
Almost give up lipo2000 now.
I'd appreciate if you can specify your protocol.


Hi there!

I tried a different transfection reagent for my CHO cells now (Perfectin from Gene therapy systems/Peqlab) and it worked a lot better (70% efficiency...) Did the transfection according to their protocol but in OptiMEM medium instead of serum containing medium .... Maybe it works for you as well...

Stardust

-stardust-