Rotiphorese

Phosphate buffered saline (PBS) pH 7.4 consisted of sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate (all AppliChem, Darmstadt, Germany). For the preparation of the 2% PAA gel, the individual components were added one after the other volumetrically to a glass beaker and mixed with a glass rod, and gel formation took place within 30 min. Reduction of ammonium persulfate (Carl Roth GmbH, Karlsruhe, Germany) by adding TEMED (Carl Roth GmbH, Karlsruhe, Germany) results in radical formation and polymerization of the acrylamide monomers. The bisacrylamide (Rotiphorese^®, Carl Roth GmbH, Karlsruhe Germany) provides crosslinking of the otherwise linear framework, resulting in a gel structure. To prepare the 0.15% HA gel, hyaluronic acid (MW 350 kDa) and agar (both Caesar & Loretz GmbH, Hilden, Germany) were each suspended separately in PBS 7.4 and boiled until a clear solution was obtained. Subsequently, both solutions were mixed and cold-stirred to room temperature. The composition of the individual hydrogels is listed in Table 1. Compared to the original HA gel, the concentration of the gel-forming components was reduced by 25% each to adjust the viscosity.

mRNA samples (1 μg) were dissolved in gel loading buffer [containing 20% glycerol in 1× TBE (Carl Roth^®)] and loaded onto a 6% polyacrylamide gel. Electrophoresis was carried out in 1× TBE (Rotiphorese^®, Carl Roth^®) buffer at 12 W for 4 h. Gels were post-stained for 20 min with Stains-all (Sigma-Aldrich) and destained overnight in 75% isopropanol. Nucleic acid bands were visualized on a Typhoon 9400 (GE Healthcare) using 633 nm. Emission signals were recorded at 670 nm.
Single-stranded siRNA samples were analyzed by denaturing PAGE. Twenty-five picomoles of oligonucleotides were loaded onto a 20% denaturing polyacrylamide gel containing 1× TBE (compounds for denaturing PAGE from Carl Roth^®). PAGE was performed in 1× TBE buffer (12 W/4 h), gels were then post-stained for 20 min with Stains-all (Sigma-Aldrich) and destained overnight in 75% isopropanol. Detection was carried out on a Typhoon 9400 (GE Healthcare), before and after staining, using 532 and 633 nm for excitation. Emission signals were recorded at settings 610BP30 nm and 670 nm.