High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale

Dicer-transfected cell pellets were resuspended in 5-pellet volumes of lysis buffer supplemented with one tablet of Roche Complete EDTA-free and incubated on ice for 10 min. The in vitro transcription of human pre-let-7a-1 with a 5'-HH ribozyme tag and a 3'-HDV ribozyme tag was carried out in 40 mM HEPES pH 8.0, 20 mM MgCl 2 , 50 mM DTT, 1 mM spermidine, 0.1% triton X-100, 4 mM of each NTP, 30 μg/mL of linearized pUC19-HH-pre -let-7a-1-HDV plasmid, 0.5 U/mL of RNasin® Plus RNase inhibitor and 30 μg/mL of His-tagged T7 polymerase prepared in house [50] . Since both ribozyme tags were cleaved off during transcription, the pre-let-7a-1 RNA was generated with homogeneous ends directly from the transcription reaction. The RNA was then purified by denaturing gel electrophoresis, as described previously [51] . To convert the purified RNA into an optimal Dicer substrate with 5′-phosphate and 3'-OH ends [52] , a phosphorylation reaction was carried out with T4 polynucleotide kinase (NEB #M0201S) according to the manufacturer protocol with either cold ATP or ATP-γ-32 P. The 32 P-labeled pre-let-7a-1 was further purified by denaturing gel electrophoresis, extracted from the gel by crush and soak, ethanol precipitated and resuspended in TE pH 7.5 (10 mM Tris pH 7.5 and 1 mM EDTA) [51] . The cold phosphorylated RNA was further purified by HPLC at 65°C on a DNApac PA-100 9 × 250 mm equilibrated with 30% DNApac-B buffer (12.5 mM Tris pH 7.4, 5 M urea and 0.5 M NaClO 4 ) and 70% DNApac-A buffer (12.5 mM Tris pH 7.4 and 5 M urea). The sample was loaded and eluted with a linear gradient (30 to 70%) of DNApac-B buffer. The fraction containing pure pre-let-7a-1 was exchanged into TE pH 7.5 and concentrated using a 3-kDa MWCO Amicon® Ultra-4 Centrifugal Filter Unit (Millipore). Both the 32 P-labeled and cold pre-let-7a-1 samples were stored at − 20°C.