proc natl acad sci u s a. 2007 july 3 104(27): 11286–11291
RNAi and Vector Transfection. RNAis were from Dharmacon (Lafayette, CO) using their RNAi design center. siControl contains at least four mismatches with all known human, mouse, and rat genes. Primary murine fibroblasts were plated at <50% confluency (300,000 cells per 60-mm plate) 16 to 20 h before transfection in penicillin/streptomycin-free medium at a ratio of 6:1 (vol:vol) Oligofectamine (Invitrogen, Carlsbad, CA) to RNAi and incubated with cells in Opti-MEM low-serum medium (Gibco BRL, Carlsbad, CA) for 5 to 6 h per the manufacturer's instructions. Expression vector pUSE-Bcl-2 was from Upstate Biotechnology (Waltham, MA). pCMV-p53 (32) and pcDNA3.1-E2F1 (33) were generous gifts of R. Reddel (Children's Medical Research Institute, Westmeade, Australia) and R. Halaban (Yale University). Lipofectamine 2000 reagent (Invitrogen) in Opti-MEM was used at a ratio of 5:1 to vector DNA per manufacturer's instructions, incubated for 4 h at 37°C, and washed. RNAi inactivation or vector expression occurred in 24 to 48 h.
Transfections with siRNA. Cells in six-well plates were transiently transfected with siRNAs with Oligofectamine (Invitrogen) according to the manufacturer's instructions. Briefly, 4 μg of siRNA against Par-4 or PKA α and β catalytic subunits or control siRNA was mixed with 175 μl of Opti-MEM (fresh RPMI medium without antibiotics) and complexed with a mixture of 3 μl of Oligofectamine and 15 μl of Opti-MEM for 20 min at room temperature. The complex was diluted to obtain a final volume of 1 ml and added to the cells. After 4 h, the cells were replenished with 500 μl of Opti-MEM containing 30% fetal calf serum and incubated for 24 h. siRNA transfections were repeated after 24 h; after a total of 48 h, the cells were processed for either apoptosis, PKA activity assays, or Western blot analysis. The siRNA for Par-4 (5′-GAUGCAAUUACACAACAGAdTdT-3′) and the siRNA for PKA catalytic subunit α (catalog number M-004649-00) and PKA catalytic subunit β (catalog number M-004650-00) were purchased from Dharmacon, Inc.
plos genet. 2006 november 2(11): e196
Drosophila SL2 cell RNAi. All RNAi for Drosophila SL2 cells was performed as described [52]. Double-stranded RNA was synthesized with T7 RiboMax (Promega, http://www.promega.com). Cells were RNAi depleted using 50 μg of dsRNA for each gene and normalized with luciferase dsRNA for co-RNAi treatments. For RNAi-transfection experiments, 15 μg of dsRNA was included in each transfection after the initial RNAi depletion. Depletion of RBF1 was confirmed by Western analysis (unpublished data). Depletion of Aac11 RNA was confirmed on microarray analysis (unpublished data).
mol cell biol. 2006 march 26(5): 1908–1916
RNA interference. Complementary 64-base short-hairpin RNA oligonucleotides were synthesized (Integrated DNA Technologies, Inc., Coralville, IA) containing a 19-base C-terminal AR sequence (5′-GCACTGCTACTCTTCAGCA-3′) in inverted repeat orientation (6). After annealing, the 64-mer duplex was inserted into BglII/HindIII-digested pH1RP RNA interference expression vector (20). After transfection of LNCaP 104-R1 cells using Effectene reagent (QIAGEN, Valencia, CA), G418-resistant colonies were selected and clones were screened for reduced AR expression by immunoblotting analysis with anti-AR polyclonal antibody (AN21) (37). All siRNA duplexes were purchased from Dharmacon Research. The siRNA targeting sequence for Bax is 5′-GACGAACUGGACAGUAACA-3′, that for for Noxa is 5′-GGAAGUCGAGUGUGCUACU-3′, and that for luciferase is 5′-CGUACGCGGAAUACUUCGA-3′. The transfection of siRNA oligonucleotides was performed with Lipofectamine 2000 (Stratagene) according to the manufacturer's recommendations.
