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).