LNP quantification and encapsulation

LNP quantification and encapsulation

Kit used: Quant-iT™ RiboGreen™ RNA Assay Kit according to manufacturer's protocol.

Preparations:

1. Dilute TEX20 buffer to X1 with nuclease free water. 
2. Prepare the aqueous Quant-iT™ RiboGreen® reagent working solution by diluting with TEx1- 200 fold for the high-range assay or 2,000-fold for the low-range assay.
3. Prepare a 2 μg/mL solution of ribosomal RNA standard (100 μg/mL) by diluting 50-fold in TEx1.
4. For the high-range standard curve, dilute the 2 μg/mL RNA solution as shown in Table 1. For the low-range standard curve, first dilute the 2 μg/mL RNA solution 20-fold into TEx1 to make a 100 ng/mL RNA stock solution, and then prepare the dilution series shown in Table 2.
5. Repeat preparation with TEx1+0.5% Triton

Table 1. High range standard curve

Table 2. Low range standard curve

Sample analysis

1. Dilute the cLNPs in TEX1 or TEX1+0.5% Triton to a final volume of 1.0 mL in eppendorfs.
2. Add 1.0 mL of the aqueous working solution of the Quant-iT™ Ribo Green® reagent (prepared above) to each sample. Incubate for 2 to 5 minutes at room temperature, protected from light.
3. Measure the fluorescence of the sample using plate reader at wavelengths 485/528.
4. Subtract the fluorescence value of the reagent blank from that of each of the samples.
5. Determine the RNA concentration of the sample from the standard curve generated in RNA Standard Curves. Compare TEx1 or TEx1+0.5% Triton diluted samples with TEx1 or TEx1+0.5% Triton diluted rRNA accordingly.
6.  Calculate % encapsulation.