I just started working with RD and RH41 rhabdosarcoma cell lines. They seem to be difficult to deal with so far. They grow very slowly.
I tried to transfect both cell lines with different concentrations of siRNA using lipofectamine RNAimax in a 6-well plate. I used 5ul of the reagent per well and left the mix of RNAiMax along with Optimum and siRNA to incubate for 15 minutes then added them to the cells with media (This is my usual protocol that I use and generally works well with other cell lines).
Having said that, after 24 hours of transfection, ALL cells were dead! including siControl treated cells whereas the cells in my untransfected well which contains no reagent were growing alright.
Does anyone have a certain protocol they have verified and used with either RH41 or RD cell line? I know the general advice would be try different lipid reagents with different volumes to cell density but I'm just wondering if someone already has a protocol that they know works for them?
Thanks!
Here's a transfection reagent optimized for siRNA transfection into RD cells (https://altogen.com/product/rd-transfection-reagent-retinal-epithelium-cells-ccl136/). The protocol is there as well, and the reagent itself is based on lipofection, which means you shouldn't encounter much cytotoxicity. If you're having problems with siRNA control, that's a sure indication that your reagent is either at too high a concentration, or that you need to try another approach like the kit above.
I usually use 100ul serum free media, 6ul of transfection reagent (usually HiPerfect from Qiagen) and 0.6 ul of 20uM siRNA solution per well.
This means a total of 106.6ul per well. Also, I put the mixed solution in a rotary incubator for 15-20 minutes before transfection.
I have tried this using different cell lines and siRNAs/transfection reagents from different companies and it remains very effective, judging by qPCR and Western Blot.
N.b, I usually perfrom the transfection after plating, although colleagues in our lab perform the first transfection 24h after plating with particularly sensitive cell lines.
I hope this helps.
Best,
Jason
Dear Sarah,
I have not a specific protocol that works on RH41 or RD cell lines, but I had the same problem as you while working with HUVECs for the first time. HUVECs are difficult to deal with and grow very slowly, as your cell lines. After 24 hours, my cells were all dead. Thus, I modified the transfection protocol. After 6 hours from transfection as described by you, I remove the medium with liposomes and siRNA, I wash the cells twice with medium without serum and I finally add cell culture medium. This protocol doesn't affect cell viability, even when working with very sensitive cells.
Best,
Francesco
Venkatesh, I decided to thaw out a new batch of cells. I figured maybe the cells were at a high passage and that maybe they were just too sensitive. I'm going to try transfecting the new batch of cells with the suggestions I got! I did hear about HiPerfect and Lipo3000 being much better to use for transfections!
With Rh4 and CW9019 (other alveolar rhabdomyosarcoma cell lines) Dharmafect 1 works beautifully.
http://www.ncbi.nlm.nih.gov/pubmed/21911456
We find high toxicity with lipofectamine 2000 as well in those rhabdo. It is not a good reagent for siRNA transfection.
Here's a transfection reagent optimized for siRNA transfection into RD cells (https://altogen.com/product/rd-transfection-reagent-retinal-epithelium-cells-ccl136/). The protocol is there as well, and the reagent itself is based on lipofection, which means you shouldn't encounter much cytotoxicity. If you're having problems with siRNA control, that's a sure indication that your reagent is either at too high a concentration, or that you need to try another approach like the kit above.
Hi Sarah,
As you know, often times it is very difficult to achieve high delivery efficiency when using a lipid based approach. May I ask if you have tried Self-Deliverable RNA silencing FANA Molecules to achieve high delivery efficiency?
If not, I encourage you to review the attached FANA delivery protocol without the use of any transfection reagents.
Please kindly let me know if this can help your specific experiments?
Have a good day!
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