The 16-nt siRNA targeting CDK9 efficiently induces endogenous gene silencing in HeLa cells. (A) The wild-type and 16-nt CDK9 siRNAs used to program RISC are shown. Arrowheads mark target cleavage sites defined by the 5′-end of the guide strand. siRNA sequences are shown in Supplemental Figure 1. (B) The 16-nt CDK9 siRNA more efficiently knocks down CDK9 mRNA than 19-nt WT siRNA. HeLa cells were transfected with 50 nM CDK9 siRNA (19-nt or 16-nt), harvested 48 h post-transfection, and 1 μg total RNA was reverse-transcribed. CDK9 mRNA levels quantified by quantitative PCR and normalized to GAPDH mRNA are presented relative to mRNA levels in mock-transfected cells. Data represent three independent experiments. (C) The 16-nt CDK9 siRNA more efficiently decreases CDK9 protein expression than 19-nt WT siRNA. Immunoblot analysis of CDK9 knockdown by 19-nt and 16-nt siRNAs. HeLa cells were transfected and harvested as in (B), and 120 μg total protein was analyzed by immunoblot using anti-CDK9 and anti-CycT1 antibodies. (D) The 16-nt CDK9 siRNA more efficiently programs RISC to cleave target CDK9 mRNA than 19-nt WT siRNA. CDK9 siRISC was programmed by transfecting HeLa cells with 19-nt or 16-nt siRNA. Target cleavage reaction was performed at 37°C for 90 min. See Materials and Methods for details. The arrow designated “32P-cap-labeled target” points to full-length 32P-cap-labeled CDK9 target mRNA. The arrows designated “cleavage product (19-nt)” and “cleavage product (16-nt)” point to products of target mRNA cleavage by CDK9 siRISC programmed with 19-nt and 16-nt siRNA, respectively.
