Silencing Assays in Mouse Hepatocytes

In vitro gene silencing assays were performed using primary mouse hepatocytes freshly isolated as previously reported (7 (link)). Free uptake and transfection assays were performed using protocols previously described (52 (link),53 (link)). Each siRNA concentration was analyzed in duplicate. For transfection assays, 5 μl siRNA at indicated concentrations were mixed with 4.9 μl of Opti-MEM (LifeTech, cat #31985–062) and 0.1 μl of Lipofectamine RNAiMax (Invitrogen, cat #13778150) per well of a 384-well plate. After incubation at room temperature for 15 min, 40 μl of William's E Medium (Life Tech, cat #A12176-01) containing ∼5×10³ primary mouse hepatocytes were added to the wells. Cells were incubated for 24 h at 37°C in 5% CO₂ prior to RNA purification. For free uptake experiments, 5 μl siRNA at indicated concentrations were mixed with 5 μl Opti-MEM (LifeTech, cat #31985-062) per well of a 384-well plate. After 15 min, 40 μl of William's E Medium (Life Tech, cat #A12176-01) or EMEM medium (ATCC) containing ∼5 × 10³ cells were added to the siRNA mixture. Cells were incubated for 24 h at 37°C in 5% CO₂ prior to RNA purification.