Does anyone have a good protocol/reagent for transfecting U937 cells with siRNA? I have used the lipofectamine method a few times now with little/no results. I purchased my siRNA material from Dharmacon (including their transfection reagent Dharmafect). I am using about 200pmol of siRNA but I still am not getting knockdown of my protein-of-interest at the 48/72h mark when validated by Western Blotting. Does anyone have any tips/advice? Or anything else that has worked better for them in the past. I have tried this technique with Mono-Mac6 as well with no luck.
On another note, I am trying to use the electroporation technique using the Amaxa Nucleoofection kit. With this technique, my cells die very quickly. They are unable to grow past 2x10^5. This kit comes with a GFP+ control, and I've noticed even at the 24h mark I lose most of that GFP. So if anyone has any advice related to that it would be greatly appreciated.
I generally use the Lipofectamine RNAiMAX kit, where I use 10-15 pmol of siRNA and 3ul of RNAi reagent. I incubate the cells for 6-8h and then provide media change. I have taken out cells for WB after 24h,36h, and 48h where at 36h I find maximum knockdown.
If it still does not work you can lodge a complaint to Dharmacon, however, I have seen Dharmacon siRNA are really good. Also, an extremely high quantity of siRNA with a lower lipofectamine reagent did not work in my case.
For transient transfection, I have used lipofectamineRNAiMAX and QIAGEN's HiPerfect Transfection reagent. Both the agents worked well for me when they are allowed to transfect the cells in media devoid of serum and antibiotics. Like Sourav Ghosh mentioned, I also allow the incubation of the transfection complex and the cells for a minimum of 4 hours (and a max of 7-8 hrs), and then I replace the media. Analysis of dose and time-dependent knockdown of my gene of interest led to a good knockdown at about 48 hrs.
You may consider the following points carefully:
1. Recheck the seeding density of your cells? It should be about 60- 70% confluent on the day of transfection.
2. Time of Incubation of siRNA and transfection reagent. Usually, it's about 10-20 minutes but you should recheck the datasheet of your reagent.
3. Recheck that you are making the transfection complex in serum-free and antibiotic-free media. (or Try Opti-MEM from Gibco specified for transfections).
4. I assume that you are doing reverse transfection so I would suggest that you can try forward transfection (in my experience I observed a comparatively higher knockdown with forward transfection though it takes one more day than the reverse transfection)
Hope it helps!!
This RNAi Kit you both mentioned, Vikrant Mehta Sourav Ghosh is it compatible with Suspension Cell Lines like U937 and Monomac6?
Kanwal Mahmood Yes it is compatible with U937 cells. However, I cannot comment on monomac6. You can visit the products site for more information.
https://www.thermofisher.com/in/en/home/brands/product-brand/lipofectamine/lipofectamine-rnaimx.html?ef_id=Cj0KCQiAys2MBhDOARIsAFf1D1eP32f-X9iEn1_oy2m5Bv4ckuG-fJSMz4Lc2AlNq8a4pIbbHKtMi0YaAkpBEALw_wcB:G:s&s_kwcid=AL!3652!3!552917619274!e!!g!!rnaimax&cid=bid_clb_tfx_r01_co_cp0000_pjt0000_bid00000_0se_gaw_bt_pur_con&gclid=Cj0KCQiAys2MBhDOARIsAFf1D1eP32f-X9iEn1_oy2m5Bv4ckuG-fJSMz4Lc2AlNq8a4pIbbHKtMi0YaAkpBEALw_wcB
Kanwal Mahmood, I looked at certain aspects of the U937 cells and found some interesting links which state that transfecting these cells is a challenging task and hence you may be finding it difficult to obtain the expected knockdown. EZBiosystems (link attached below) are having some proprietary transfection reagent suitable for these cells which you may try.
http://ezbiosystems.com/view2.asp?d_id=184&Name=U-937%20Cell%20Avalanche%99%20Transfection%20Reagent%20(human%20lymphoma%20cell)
Other than that there is some other link that may be helpful https://www.mirusbio.com/tech-resources/tips/different-types-of-transfection-protocols.
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