Has anyone tried Accell siRNA (Dharmacon) to knockdown gene expression in primary human CD4+ T cells? I have found one paper where this was done (http://jem.rupress.org/content/209/10/1743.full#F4) but would be grateful if others could share their experience using this reagent. I'm particularly concerned about the need to culture for >48 hours in serum-free conditions and how this may affect primary T cells.
Many thanks.
The vendor of silencing RNA duplexes is not so important as the target sequence. In fact, this vendor actually is considered one of the best in the market. I'm not familiar with silencing genes in primary human cells but it's important to have proper positive and negative controls because siRNA transfection and concentration among many other variables can influence the outcome. Having a positive control to work would validate those other variables. Good luck.
I have tried many siRNA knockdown approaches, all generally work but you get a high level of cell death. If you are culturing for longer than 24 hours you will need to use a lentivirus. Alternatively, we are using locked nucleic acids and are finding that they work very well, although they can be expensive.
Thanks for your answers! Adam, I'm not familiar with using locked nucleic acids for gene knock down but it sounds interesting - would you be able to tell me a little more about how you do this in T cells?
Locked nucleic acids are great, we have been using then for a few years now. You can order and design them from exiqon. You basically just add them to the culture media without any transfection reagents and then 6 days later you have a knockdown. Here is a paper http://nar.oxfordjournals.org/content/33/1/439.full.pdf+html then explains it better.
Hi Ceri
I have used Accell siRNAs for silencing of gene expression in primary human CD3+ T cells (pre-activated with PHA and IL-2). I incubate the cells (2 x 10(6) cells/ml in Accell delivery medium for 72 hours with 1uM siRNA and routinely achieve >80% gene silencing at the protein level. The cells are fine in the Accell delivery medium. I have not tried non-activated T cells yet but it should be ok. 1uM Accell siRNA is recommended by Dharmacon but you can titrate this down (will probably depend on your target of interest). If you need any more information drop me a message.
Best Wishes
Mike
We use alternative self-deliverable RNAi (sdRNAi) technology. It has similar characteristic to Accel (no transfection reagents), but works in 3-5% serum. We successfully used it against multiple T-cell targets, including PD1, cbl-b, TIGIT to name a few. Will be happy to discuss
HI Michael,
have you had a chance to try these siRNA in resting (non activated) T cells yet?
Hi Dequina.
No I have only used activated T-cells for knockdown experiments using Accell siRNAs. There are a few publications that have used non-activated T-cells using this technology. If you Google 'T cells and Accell siRNA' they should appear, albeit you will likely need to scroll through 5-6 pages to get them.
All the best
Mike
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