Hi Kim Sung

I have some suggestions regarding your problem:

1. Test Multiple Ratios: Experiment with different RNA:lipid ratios (e.g., 1:1, 1:2, 1:3, or 1:4). Excess Lipofectamine may increase toxicity, while insufficient amounts may lower efficiency.

2. For RNAi, a starting point is 5-20 pmol RNAi per 24-well plate with 1-2 µL Lipofectamine 2000.

3. Use a validated siRNA or ASO targeting a highly expressed housekeeping gene (e.g., GAPDH) or a fluorescent reporter siRNA (e.g., Alexa Fluor-labeled siRNA) to confirm the delivery is efficient.

4. If knockdown is still absent in the positive control, focus on improving transfection conditions rather than the RNAi/ASO design.

Optimize the Transfection Procedure:

1. Serum-Free Media: Use Opti-MEM or another serum-free medium during complex formation, as serum can interfere with complex assembly.

2. Incubation Time for Complex Formation: Allow the RNAi/ASO and Lipofectamine 2000 to incubate for exactly 15-20 minutes before adding to cells.

3. No Antibiotics: Avoid antibiotics during transfection as they may interfere with cell health and efficiency.

Thank you for your response!

I will test with multiple ratios.

Dear Kim,

The recommendations made by Hossein are very interesting and should be definitively followed.

However, in case the gene silencing is still not enough I would like to propose you 2 options to increase the overall efficiency:

1. Increase L2000 capacity using CombiMag. CombiMag is a magnetic nanoparticles formulation that we have developed specifically to combine with the already existing transfection reagent, to increase their efficiency with the help of Magnetofection***.

In this way the original protocol is not modified, CombiMag is simply added at a ratio of 1:1 with nucleic acid to improve transfection.

2. Use an alternative to L2000. In parallel to Magnetofection we propose also “classical” transfection reagents and one of ours is Lullaby that was specifically developed for siRNA transfection.

It is totally biodegradable and thus almost not toxic and really efficient as demonstrated by the numerous publications.

I would like to cite the article from the Cancer Research UK Beatson Institute, in which the author stated :"Often we find that reagents suggested by collaborators are effective at transfecting siRNA but demonstrate associated toxicity at the concentrations we require. By far, our preferred reagent is Lullaby from OZ Biosciences. We have used this reagent in over 20 cell lines and have found it essential in enabling siRNA screens in hard to transfect cell lines such as those derived from GEMM pancreatic tumours and AML suspension cell lines, with minimal toxicity."

Moreover Lullaby was demonstrated to be efficient in MiaPaCa-2 (Meng D. EMBO J . 2021 May 14;e106412. doi: 10.15252/embj.2020106412.) and PANC-1 (Papalazarou V. J Cell Sci. 2022 Dec 15;135(24):jcs259978.).

you can have more infos at: https://ozbiosciences.com/cell-lines/24-203-lullaby-sirna-transfection-reagent.html#/12-capacity-500_l

Would you like to give CombiMag or Lullaby a try, please do not hesitate to contact me via ResearchGate or directly at: [email protected]; I will be glad to send you a sample !

Good luck with your experiment,

best regards,

Cedric

*** The Magnetofection technology uses a magnetic field to attract and concentrate complexes of magnetic nanoparticles and nucleic acid onto the cell surface as demonstrated by Grześkowiak BF et al, Pharm Res. 2014 Jul 18. (http://www.ncbi.nlm.nih.gov/pubmed/25033763).