Nucleic acid detection kit

cRNA probes for Tlr8 were synthesized in the presence of digoxigenin (DIG)-labelled UTP using a DIG RNA Labelling Kit in accordance with the manufacturer's protocol (Roche Diagnostics, Mannheim, Germany). The primer pairs for each probe are shown in Table 3 (product size: 758 bp). Cryosections (6 μm) were treated with acetylation solution and digested with proteinase K. The sections were incubated with a prehybridization solution and then with a hybridization buffer containing 50% formamide, 10 mM Tris-HCl (pH 7.6), 200 mg/mL RNA, 1× Denhardt's solution (0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and 0.02% Ficoll PM400; Sigma-Aldrich, St. Louis, MO, USA), 10% dextran sulphate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA (pH 8.0), and a sense or antisense RNA probe (final concentration, 0.2 μg/mL) for 24 h at 58°C. After washes in saline sodium citrate buffer, the sections were then incubated with 0.2% polyclonal sheep anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (1: 400; Nucleic Acid Detection Kit, Roche Diagnostics) for 24 h at room temperature. The signal was detected by incubating the sections with a colour substrate solution (Roche Diagnostics) containing nitro blue tetrazolium/X-phosphate in a solution composed of 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl₂ in a dark room overnight at room temperature.

Total cell RNA and mitochondrial RNA were extracted using the TRIzol^® method followed by column purification (Direct-zol™ RNA MiniPrep; Zymo Research, Irvine, CA). mRNA was further purified by polyA selection using a Dynabeads^® mRNA Purification Kit (Thermo Scientific, Waltham, MA). The RNA concentration and purity were checked using a NanoDrop™ (Thermo Scientific) and agarose gel electrophoresis. Then, 130 ng of each RNA sample was separated on a 10% precast polyacrylamide tris-borate-EDTA(TBE)-urea gel (Bio-Rad, Hercules, CA) and transferred onto a positively charged nylon transfer membrane (Ambion^®; Life Technologies). The blot was hybridized with 200 ng/mL digoxigenin (DIG)-labeled SHLP6 RNA probe in Easy Hyb™ hybridization buffer (Roche) for 16 h at 60°C. After two stringent washes in 2× SSC (saline sodium citrate buffer) (1× SSC contains 0.15 M NaCl and 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) for 5 min at room temperature and 0.2× SSC-0.1% SDS for 15 min at 60°C, the membrane was incubated for 30 min at room temperature with an anti-DIG antibody conjugated to alkaline phosphatase (Roche). Subsequent visualization was achieved using the BCIP-NBT substrate as instructed using a nucleic acid detection kit (Roche).