The cytotoxicity of RNA nanoparticles can be evaluated with an MTT assay kit (Promega) following the manufacturer’s instruction. Briefly:

1. Design your experiment setting with the control groups, concentrations, and technical repeats. Prepare the cells in 96-well plate by seeding them at about 40% confluency, and culture at 37°C incubator with humidified air containing 5% CO2 overnight.

2. Incubate cells with RNA nanoparticles in complete cell culture medium for 48-72 hours: suspend the RNA nanoparticles in fresh cell culture medium with a 10% fetal bovine serum (FBS) at the indicated concentrations and add to the cells for incubation at 37°C for 48-72 hours.

3. Add 15 uL of MTT dye solution to each well and incubate at 37°C for 4 h; add 100 uL of solubilization/stop solution from the MTT assay kit to each well and incubated at room temperature for 2 h for color development.

4. Record the absorbance at 570 nm using a microplate reader (Synergy 4; BioTek Instruments, Inc.). Calculate the cell viability relative to the absorbance of cells in the control group without RNA nanoparticles treatment (viability of cells in only control = 100%).

Reference:

1. Pi F, Binzel D, Lee TJ, Li Z, Sun M, Rychahou P, Li H, Haque F, Wang S, Croce CM, Guo B, Evers BM, Guo P. Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression. Nature Nanotechnology. 2017. doi:10.1038/s41565-017-0012-z. 

2. Li H, Rychahou PG, Cui Z, Pi F, Evers BM, Shu D, Guo P, Luo W.?RNA Nanoparticles Derived from Three-Way Junction of Phi29 Motor pRNA Are Resistant to I-125 and Cs-131 Radiation. Nucleic Acid Ther. 2015 July 20;25(4):188-197. 

3. Pi F, Zhang H, Li H, Thiviyanathan V, Gorenstein DG, Sood AK,?Guo P. RNA Nanoparticles Harboring Annexin A2 Aptamer Can Target Ovarian Cancer for Tumor-Specific Doxorubicin Delivery. Nanomedicine. 2017 Apr;13(3):1183-1193.