Cytoplasmic RNA isolation and immunoprecipitation of dsRNA

Please find below the protocol for Cytoplasmic RNA isolation and immunoprecipitation of dsRNA below: 

Cytoplasmic RNA was extracted from mouse olfactory epithelium/mouse olfactory bulb/50mg specified region of postmortem human brain using RNeasy kit (Qiagen) except that the standard lysis buffer was exchanged for the lysis buffer RNL [50 mM Tris–HCl, pH 8.0; 140 nM NaCl; 1.5 mM MgCl2; 0.5% (v/v) Nonidet P-40 (1.06 g/ml); and 1 mM DTT added just before use]. Purified RNA was immunoprecipitated using a dsRNA-specific antibody (J2, Scicons) in 200 μl binding buffer (0.025% Triton X-100 in PBS). with 5 μg of J2 antibody and 1 μl RNaseOUT (Life Technologies) rotating overnight at 4°C. J2- bound dsRNA was incubated in binding buffer with 50 μL of prewashed protein-A or G Dynabeads beads for overnight at 4°C, (Life Technologies) followed by 5× washes in cold binding buffer. RNA was then extracted with TRIzol Reagent as above. cDNA was synthesized with SuperScript™ III Reverse Transcriptase (Thermo Fisher). 

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