siRNA ADAM17

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Publication protocol

RNAi was used to specifically knock down ADAM17 (AAI46659) expression in Bosc23 cells. Small interfering RNAs (siRNAs) were expressed from short hairpin RNAs (shRNAs) using the MISSION™ shRNA (Sigma) plasmids TRCN0000052168 to TRCN0000052172. Cells were co-transfected with plasmid encoding ShhNp cDNA and shRNA plasmids or empty control plasmid, and transfected cells were enriched by the addition of 5 μg/ml puromycin for 30 h before starting the assay. In addition, to confirm findings obtained by shRNAi, ADAM17 was knocked down in ShhNp-expressing Bosc23 cells by using 5 nmol of On-Target plus anti-human ADAM17 pooled siRNA (Dharmacon) using the siGENOME nontargeting siRNA pool as a control. In all experiments, the amount of ShhNp released into the medium was quantified using ImageJ. Data were plotted as released versus cell-bound ShhNp relative to the value obtained with the empty vector control, which was set to 100%. Statistical analysis was performed in Excel using Student's t test (two-tailed, unpaired). All values shown throughout are ±S.D. To assess the effectiveness of ADAM knockdown, semiquantitative RT-PCR analysis of ADAM17 and glypican 1 (Glp1) expression in Bosc23 cells transfected with pooled siRNAs specifically interfering with ADAM17 mRNA was conducted. PCR products were quantified using ImageJ, and their relative intensities were calculated. Semiquantitative RT-PCR revealed a 60% reduction in ADAM17 mRNA levels in Bosc23 cells cotransfected with five pooled shRNAs specific for the protease. As a control, glypican 1 mRNA levels were not affected by ADAM17-specific mRNA knockdown.

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