Protocols

Cell Assay Protocol 001: Flow Cytometry to Test the Binding of RNA Nanoparticles with Cells

1. Culture cells in their complete medium (growth medium with 10% FBS). Dissociate the cells with trypsin and wash with PBS once.

2. Incubate 0.2 million cells with fluorescently labeled RNA nanoparticles at a final concentration of 50 nM to 100 nM in their growth medium, at 37 °C for 1 to 2 hrs.

3. Wash cells with PBS, re-suspend cells in 200-300 ul of PBS buffer.

4. Assay the binding of RNA nanoparticles with cells by Flow Cytometry, decide excitation and mission spectrum based on the information from your product.

Reference:
1. Shu Y, Shu D, Haque F, Guo P. Fabrication of pRNA Nanoparticles to Deliver Therapeutic RNAs and Bioactive Compounds into Tumor Cells. Nature Protocols 2013;8:1635-1659. [link]

2. 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; 9:9731-9740. [link]

3. Shu D, Shu Y, Haque F, Abdelmawla S, Guo P. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology 2011; 6:658-67. [link]

Cell Assay Protocol 002: Confocal Microscopy to Test the Internalization of RNA Nanoparticles within Cells

1. Culture cells on cover glass in their complete medium overnight.

2. Wash cells in growth medium without FBS twice before the experiment. Incubate cells in each well with fluorescent RNA nanoparticles at a final concentration of 50 nM to 200 nM at 37 °C for 1 to 4 hours; wash cells with PBS.

3. Fix cells with 4% formaldehyde in PBS at room temperature for 20 minutes, then wash cells with PBS.

4. Stain cells with a cytoskeleton staining reagent or membrane staining reagent, then wash cells with PBS.

5. Mount the cover glass with cells onto a glass slide, and stain the cell nuclei with DAPI. After drying, the slides are ready for confocal microscopy analysis. Use excitation and emission wavelength for equipment setting based on the information from your product.

Reference:

1.   Haque F, Shu D, Shu Y, Shlyakhtenko L, Rychahou P, Evers M, Guo P. Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers. Nano Today. 2012; 7(4):245-57. [link]

2.   Khisamutdinov EF, Li H, Jasinski DL, Chen J, Fu J, Guo P. Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles. Nucleic Acids Research 2014; 42(15):9996-10004. [link]

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 2016 Nov 25. doi: 10.1016/j.nano.2016.11.015. [link]

Cell Assay Protocol 003: MTT Assay to Test the Effect of RNA Nanoparticles within Cells

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. [link]

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. [link]

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. [link]

Cell Assay Protocol 004: Dual Luciferase Assay to Test the Cellular Delivery of anti-microRNA by RNA Nanoparticles

1. Culture cells in 24-well plate in their complete growth medium with 10% FBS overnight.

2. When the cells reach 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 the experiment. Incubate cells with anti-microRNA harboring RNA nanoparticles at a 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 the Dual Luciferase® Reporter Assay system (Promega, E1091). Following the instruction to lyse cells and read the firefly luciferase and Renilla luciferase signal with the microplate 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. [link]
  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. [link]

Animal Experiment Protocol 001: Test the In Vivo Targeting of RNA Nanoparticles in Mice

This is a single dose animal experiment to briefly test the biodistribution property of the fluorescently labeled RNA nanoparticles in mice.

1. Prepare the mice model with tumor xenograft of your choice.

2. Following your ICAUC instruction conditions, inject sterile fluorescent RNA Nanoparticles in PBS into mice through the tail vein. Usually injection around 10 nmol of fluorescent RNA nanoparticles in 100 µl of PBS per mice.

3. Monitor the biodistribution of fluorescent RNA Nanoparticles in the mice using IVIS® Lumina station (or equivalent) at different time point post injection. Choose the filter settings for the imaging equipment referring to your RNA nanoparticles information.

4. Euthanize the mice after the nanoparticle distribution reached an equilibration phase, usually after 6-10 hours. Take out the organs and tumors for whole body imaging to visualize the accumulation of RNA nanoparticles.

5. The tumor or organ of interest can be retained for further microscopic study. Fixing the organ in tissue fixation buffer (10% sucrose, 4% formaldehyde in PBS) at 4°C overnight. After fixation, the tissue can be cryosectioned for further analysis using confocal microscopy.

Reference:
1. Haque F, Guo P. Overview of methods in RNA nanotechnology: synthesis, purification, and characterization of RNA nanoparticles. Methods in Molecular Biology 2015; 1297:1-19. [link]
2. Shu D, Shu Y, Haque F, Abdelmawla S, Guo P. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology 2011; 6(10):658-67. [link]
3. Li H, Zhang K, Pi F, Guo S, Shlyakhtenko L, Chiu W, Shu D, Guo P. Controllable Self-Assembly of RNA Tetrahedrons with Precise Shape and Size for Cancer Targeting. Advanced Materrials 2016; 28(34):7501-7. [link]
4. Rychahou P, Haque F, Shu Y, Zaytseva Y, Weiss HL, Lee EY, Mustain W, Valentino J, Guo P, Evers BM. Delivery of RNA Nanoparticles into Colorectal Cancer Metastases Following Systemic Administration. ACS Nano 2015; 9(2):1108-16. [link]

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