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siRNA knockdown in A549

MO Miho Oka
LX Liu Xu
TS Toshihiro Suzuki
TY Toshiaki Yoshikawa
HS Hiromi Sakamoto
HU Hayato Uemura
AY Akiyasu C. Yoshizawa
YS Yutaka Suzuki
TN Tetsuya Nakatsura
YI Yasushi Ishihama
AS Ayako Suzuki
MS Masahide Seki
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Reverse siRNA transfections were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and siRNA sets (Additional file 10: Table S10). siRNA for UPF1 was kindly provided by Dr. R. Onoguchi-Mizutani and Dr. N. Akimitsu from The University of Tokyo [45]. We applied 75 pmol of siRNA for UPF1 knockdown and mixed two different siRNAs (12.5 pmol each) for SF3B1. Lipofectamine RNAiMAX (7.5 μl) and each siRNA were diluted in 400 μl of OptiMEM, then incubated for 20 min at room temperature. We seeded 1.2 × 105 cells of A549 in antibiotic-free DMEM into each well of a 6-well plate. After incubation, we added 400 μl of the RNAiMAX and siRNA mixture to each well. After 24 h, we replenished the medium and re-added the RNAiMAX and siRNA mixture. Cells were collected 48 h after the last transfection.

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