Low range ssrna ladder

To confirm the integrity of Alveofact-coated polyplexes after sonication, a gel retardation assay was performed. A 1% Agarose gel was prepared and stained with ROTI®GelStain Red. Polyplexes were prepared with 300 pmol siRNA at three different Alveofact:PEI ratios (0, 2.5:1 and 5:1). As positive control, polyplexes were treated with 1 USP unit of heparin. 3 μL of low range ssRNA ladder (New England BioLabs, Ipswich, Massachusetts, USA) and 3 μL of siRNA were respectively diluted in 27 μL of RNA free water. 30 μL of each sample were mixed with 5 μL of loading dye (New England BioLabs, Ipswich, Massachusetts, USA), loaded into the slots of a gel, and electrophoresis was run at constant voltage of 200 V for 15 min in Tris-borate EDTA buffer. The gel was visualized using a ChemiDoc MP imaging system (Bio Rad, Hercules, California, USA).

The RNA samples were thermally annealed in a folding buffer containing
0.5× TE buffer with 1 mM MgCl₂ and 100 mM NaCl. For
each nanostructure, 100 nM of ssRNA in folding buffer was rapidly
folded by heating to 80 °C for 5 min followed by cooling to 20
°C at 0.1 °C/s. The samples were run in a 5% native PAGE
gel in an ice bath along with a low-range ssRNA ladder (New England
Biolabs, Inc.). The gels were prepared using acrylamide/bis-acrylamide
(29:1) containing 1 mM MgCl₂ and 100 mM NaCl. The gels
were post-electrophoresis stained with SyBr Green (ThermoFisher Scientific)
for 15
min and imaged using Bio-Rad Gel Doc XR system.

A standard 12% acrylamide gel [1X TBE, 12% acrylamide (19:1 acryl:bis-acryl)] was used to separate the plant sRNA. The gel was warmed to ~50 °C by running it for 10–15 minutes at 40 V in 1X TBE running buffer. 1 μg of sRNA was combined with an equal volume of Gel Loading Buffer II and then used for loading the gel. The gel was run until the leading dye travels about 4–5 cm down the gel, at 70 V. Gel was stained with SYBR Gold (Life Technologies) according to manufacturer’s extraction. Different gel slices (covering the range from 10 to 60 nt, 70–100 nt and 100–150 nt), were recovered according to the band profile and the two ladders used (the Low Range ssRNA Ladder and the microRNA Marker, both from New England BioLabs inc.) and crushed in 1 M NaCl and incubated overnight at 4 °C. The day after, the samples were centrifuged for 5 min at 2000 g and the supernatant transferred to a 50 ml tube. Crushed gel slices were re-elute with 2 ml of 1 M NaCl for 1 hr at room temperature and centrifuged again. The pooled supernatants were purified with MEGAclear Kit (Life Technologies). Obtained fractions were analyzed using the Agilent Small RNA kit, to check the sizing and purity of the fractionation performed as well as the miRNA (21 nucleotides) content, according to the manufacturer’s software.