To begin mix 0.1 molar sodium acetate and 0.1 molar acetic acid in deionized water at pH 4.5. Then prepare 100 millimolar sodium sulfate buffer in deionized water at room temperature. Weigh the commercialized chitosan to prepare a 0.02%weight by volume solution and dissolve it in the prepared 100 millimolar sodium acetate buffer.
Then dissolve 20 micrograms of double stranded RNA or dsRNA in 50 microliters of nuclease free water. Add this solution to 50 microliters of the sodium sulfate buffer to make a 100 microliter dsRNA solution. Next, add 100 microliters of the prepared chitosan solution to 100 microliters each of the dsRNA solution and 50 millimolar sodium sulfate.
After mixing, heat the solutions at 55 degrees Celsius for one minute. Immediately vortex the mixture at high speed for 30 seconds to facilitate nanoparticle formation. Centrifuge the mixture at 13, 000 G at room temperature for 10 minutes to obtain a white pellet.
Transfer the supernatant to a fresh 1.5 milliliter tube and let the pellet air dry at room temperature for 10 minutes. Using a micro volume spectrophotometer, determine the concentration of dsRNA in the supernatant. To calculate the percentage of dsRNA encapsulated, divide the amount remaining in the supernatant by the initial amount of dsRNA.
