2.2 NC formation
20 μM of siRNA, 100 μM FITC-siRNA, or 100 μM Cy3-siRNA were diluted in NFW, and 600 μM peptides were dissolved in DPBS. All solutions were stored at -80oC until use. The fixed amount of siRNA was incubated with a fusion peptides solution of a nitrogen/phosphate ratio to proper volume filled with DPBS as described below, at room temperature for 30 minutes. The nitrogen/phosphate (N/P) ratio was calculated from nitrogens of fusion peptides divided by phosphates of siRNAs. First, the formation of NCs was observed using gel retardation assay. 10 pmol of siRNA was incubated with RS peptide of N/P ratio (0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 3, and 5) and RT peptide of N/P ratio (1, 5, 10, 20, 30, 40, 50, 60, and 70) in final volume of 10 µL filled with DPBS as described above. After the addition of 6× loading dye, mixed solutions were loaded to wells of 1% (w/v) agarose gel in 1× TAE buffer (40 mM tris, 20 mM acetic acid, 1 mM EDTA, pH = 8.6). Electrophoresis was run at 100 V for 30 minutes using the Mupid-2plus electrophoresis system (Optima Inc., Tokyo, Japan). TopRed stained RNA bands were visualized using the ChemiDoc™ XRS + System (Bio-Rad, CA, USA).