RNA native and denaturing agarose gel electrophoresis

RNA native agarose gel electrophoresis was performed as described previously with a few modifications (Skripkin et al., 1994). For sphere-forming and network-forming RNAs, RNAs were diluted into 4 µl buffer A (150 mM NaCl, 25 mM Tris-Cl, pH 7.4) to a final concentration of 5 µM (CLCA2 1.7 µg/µl, TLR8 1.6 µg/µl, HNRNPH3 1.9 µg/µl, LHFPL6 1.7 µg/µl, TNFSF11 1.8 µg/µl, TSPAN13 1.7 µg/µl). RNAs were incubated at 95°C for 2 min in a PCR machine and then incubated on ice for 2 min. RNAs were kept at 37°C for 2 hr. Also, 1 µl native agarose gel loading buffer (6× stock: 60% glycerol, 10 mM Tris-Cl, pH 7.4, 0.03% bromophenol blue, and 0.03% xylene cyanol FF) was added into the RNA. A total of 1 µg RNA was loaded into the 1% agarose gel made with the Tris-acetate-EDTA (TAE) buffer for electrophoresis with TAE buffer.

For RNAs containing dimerization elements (TLR8 3′UTR, MYC 3′UTR, D1a-TLR8-D2, D1b-MYC-D2), each RNA was diluted into 4 µl buffer A (150 mM NaCl, 25 mM Tris-Cl, pH 7.4) to a final concentration of 2 µM. RNAs were incubated at 95°C for 2 min in a PCR machine and then incubated on ice for 2 min. Also, 2 µl TLR8 3′UTR or 2 µl MYC 3′UTR were each diluted with 2 µl buffer A. A 2 µl TLR8 3′UTR and 2 µl MYC 3′UTR were mixed together. Then, 2 µl D1a-TLR8-D2 or 2 µl D1b-MYC-D2 were each diluted with 2 µl buffer A. Also, 2 µl D1a-TLR8-D2 and 2 µl D1b-MYC-D2 were mixed together. The final concentration of each RNA was 1 µM (TLR8 326 ng/µl, MYC 154 ng/µl, D1a-TLR8-D2 345 ng/µl, D1b-MYC-D2 193 ng/µl) in 4 µl buffer A. RNAs were kept at 37°C for 2 hr. A 1 µl native agarose gel loading buffer was added to the RNA. A total of 1 µg RNA was loaded onto the 2% agarose gel made with the Tris-borate-EDTA (TBE) buffer for electrophoresis with TBE buffer.

For denaturing agarose gel electrophoresis, glyoxal was used. RNAs were mixed with 10 µl glyoxal and incubated at 55°C for 60 min and then incubated on ice for 10 min. A 2 µl agarose gel loading buffer was added into the RNA. A total of 1 µg RNA was loaded into the 1% agarose gel made with the TAE buffer for electrophoresis with TAE buffer.