4.7. Biotinylated RNA Pull-Down Assay

To prepare the biotin-labeled RNA, 2 ug plasmid pcDNA3.1 α1AT were first linearized with NheI, purified and then transcripted by T7 RNA polymerase (NEB, Ipswich, MA, USA) with Biotin-RNA labelling Mix (Roche, Basel, Switzerland). The Biotin labelled RNA was then digested with RNase-free DNase and purified with RNA Cleanup Kit (Qiagen, Hilden, Germany). For biothylated RNA pull down, C3A cells were first lysed by extraction buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5% glycerol, 0.1% Triton X-100, 1 mm DTT, 1 mm PMSF and protease Inhibitor) the nuclear and debris were pelleted by centrifugation at 13,000 rpm for 20 min. In each assay 2 μg of biotinylated transcript were introduced into 500 ug of C3A cell extract incubated and shaking in 4 °C for 1 h; 20 μL of washed Streptavidin agarose beads (Invitrogen, Carlsbad, CA, USA) were added to each binding reaction and further incubated at RT for 1 h. Beads were then washed briefly three times with lysis buffer and collected by centrifugation, and then were loaded on SDS-PAGE gels, and the corresponding band was sent for mass spectrometry analysis.