I was wondering if someone has optimised a protocol for co-transfection of DNA and siRNA in Hela cells ? I am attempting a rescue experiment were siRNA treated Hela cells are transfected with the corresponding cDNA. However, it results in cytotoxicity.
Hi Amit,
I have performed similar rescue experiments, however I typically do a sequential transfection in which I knockdown my gene of interest with siRNAs using hyperfect for 24hrs. Followed by transfection of my expression plasmid with lipofectamine for an additional 24-48 hrs. This approach worked well for me. However, you can do co-transfection using lipofectamine 2000 reagent. Good luck!
Hi Amit, I recommend DreamFect Gold for this application. Attached is a specific protocols for cotransfection. For Hela we also have specific procedure
In addition here are few publications:
http://www.ncbi.nlm.nih.gov/pubmed/21857679
http://www.ncbi.nlm.nih.gov/pubmed/21930711
http://www.ncbi.nlm.nih.gov/pubmed/20339350
I hope this can help
I have done this frequently and I prefer to use nucleofection from Amaxa as I can simultaneously transfect plasmid and siRNA with good efficiency and transfect lots of cells at once. In Hela cells I would use kit R and protocol A028 or I013 which gives you good efficiency and not much cell death. Link to the Amaxa nucleofection supplier: http://bio.lonza.com
Thanks to all for suggestions. I will try Amaxa and Viromer green for my experiments.
Dear Amit,
I would like to let you know that since the post of your question in February, there is a new reagent that was launched and that is dedicated to HeLa transfection; HeLaFect
it is really efficient, non-toxic and presents a high compaction level authorizing co-transfections from DNA-DNA to siRNA-DNA co transfections. Moreover as it is based on biodegradable properties, you can easily perform sequentiel transfections as you has planned, with extremely low toxicity.
You can find more information on HeLaFect at : http://www.ozbiosciences.com/transfection-cell-specific/77-hela-cells-transfection-helafect-reagent.html
I would be more than glad to offer you samples to test,
would you have any question, please do not hesitate to contact me at: [email protected]
good luck with your experiment,
best regards,
Cedric
Thanks Cédric Sapet for information. I will surely try HeLaFect reagent.
Lipofectamine 2000 is perfect, we used it for several years to deliver Silencer Select and other siRNA + plasmid into HeLa and other cells
Lipofectamine should be adequate for cells easy to transfect. For primary cells (not easy to transfect), I would also propose to use electroporation via AMAXA techniques.
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