Hi Cory, it depends on your cell type and on the transfection. Are you using a cell line or a primary cell? I have done co-transfection with siRNA and plasmid in a T-and B cell line using electroporation. I just mix the plasmid and siRNA before adding it to the cells, incubate it for 15 min at RT i the cuvette before electroporation. Does your plasmid have a tag that can be detected by Flow?

-Ingvild

Hello Cory,

my experience ist that e.g. Lipofectamne 2000 works very good for transfecting both, siRNA and plasmids. I guess your cell cycle phenotype needs a while so transfecting both a the same time might be a good starting point. Probably you have to fiddle about a bit.

cheers

Michael

Depending on the transfection reagent you are planning to use, I think you should be able to co-transfect your cells with both the plasmid as well as the siRNA. Lipofectamine 2000 allows such transfections, but you will need to optimize for your specific conditions. I have attached the recommended protocol given by Life Technologies using Lipofectamine 2000. Hope it helps!...

http://www.lifetechnologies.com/uk/en/home/references/protocols/cell-culture/transfection-protocol/plasmid-co-transfection-protocol-lipofectamine-sirna.html

This is great, thanks for your responses. The cell lines I'm using are HeLa cells and RPE cells, but I'm focussing more on the Helas for now. Thanks again everyone.

Now to make this technically more difficult. After I knock-down my target gene (a mitotic kinase), I see the best mitotic phenotype 72 hr post transfection. To ensure expression of the plasmid siRNA resistant kinase, should I just transfect more to ensure expression for up to 72 hours?

Hey Cory. I think if you are able to transiently express the plasmid in your HeLa cell lines, you should be able to see expression upto 72 hours. I think all transiently transfected plasmids can be expressed for 48-72 hours, depending on culture conditions and cell lines.

Hi, I've done rescue experiments in different epithelial cell lines and I normally see good rescue after 48h. I'd just check the expression of your protein at different days up to 72h after the transfection to see how long it persists and you can titrate the amount you transfect to avoid problems of toxicity due to very high expression.

Hi Ocosio,

You can use lipofectamine 2000 for co-transfections of your plasmid and siRNA however if you could optimise your plasmid expression and siRNA knockdown day you don't need to do them together. For example, I use Huh7 to transfect my plasmid and it gives good expression after 3 days, on day 2 i transfect my siRNA and it gives good knockdown after 1 day so at day 3 i harvest my cells. That way I avoid co-transfection because siRNA efficacy decreases after 2 days and it won't be a good idea if you do co-transfection of your plasmid and siRNA.

In my case, I see the best phenotype of my kinase knockdown on day three, not when I harvest, fix and stain on day 2. This is mostly due to the function. The kinases has an adverse effect on the cell cycle, which leads to death ultimately and I think once the siRNA gets in the cell and depletes the kinasen the fate of the cell is determined early on but manifests the phenotype on day 3.

Abbas, which transfection reagent did you use to transfect plasmid then siRNA??

Dear Ocasio,

When I do them separately I prefer PEI for plasmid transfection and RNAi lipofectamine for siRNA. You can also use lipofectamine2000 for both, however, PEI is less toxic so I normally do it this way.

Hello. If I want to use ips cells and co-transfected siRNA and my DNA plasmid. What do you think that it would be the best protocol?

Thank you