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

1. Prepare the diseased mice model of your choice.
2. Inject sterile fluorescent RNA Nanoparticles in PBS into mice through tail vein, under the condition following your ICAUC instruction. Usually injection around 5-10 nmol of fluorescent RNA nanoparticles in 100 µl of PBS per mice.
3. Monitor the distribution of fluorescent RNA Nanoparticles in the mice using IVIS® Lumina station (or equivalent), choose the filter setting 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 sectioned 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.
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.
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.
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.
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