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Cell Assay Protocol 003: Dual Luciferase Assay to Test the Cellular Delivery of anti-microRNA by RNA Nanoparticles

1. Culture cells in 24-well plate in their complete medium overnight.
2. When the cells reached 80% confluence, transfect 150 ng of Psi-Check2 (Promega) or Psi-Check2 plasmid containing an oncogenic microRNA binding sequence at the 3'-UTR Region of Renilla Luciferase gene using lipofectamine 2000 (Life Technologies); after 4 hours, change the medium to complete medium and incubate with cells for another 2 hours.
3. Wash cells in blank medium twice before experiment. Incubate cells with anti-microRNA harboring RNA nanoparticles at various final concentration of 50 nM to 200 nM in opti-MEM (Life Technologies) at 37 °C for 2 to 4 hrs, then add complete medium to grow for 24 hours.
4. Test the oncogenic microRNA expression level with Dual Luciferase® Reporter Assay system (Promega, E1091). Following the instruction to lyse cells and read the firefly luciferase and Renilla luciferase signal with micro plate reader. The relative ratio of Renilla against Firefly luciferase is reversely correlated to the expression level of microRNA to be tested.

Reference:
1. Shu D, Li H, Shu Y, Xiong G, Carson WE, Haque F, Xu R, Guo P. Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology. ACS Nano 2015; 9(10):9731-9740.
2. Binzel DW, Shu Y, Li H, Sun M, Zhang Q, Shu D, Guo B, Guo P. Specific Delivery of MiRNA for High Efficient Inhibition of Prostate Cancer by RNA Nanotechnology. Molecular Therapy 2016; 24(7):1267-77.
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