2.12. SiRNA Transfection to Knock down Pim-2

The MM cells were inoculated into 6-well plates, and approximately 2 mL of antibiotic-free medium was added to each well so that the cell density at the time of transfection was between 40% and 60%. Next, 4 μL/well of the Lipo 3000 transfection reagent was diluted with 200 μL of serum-free medium, mixed, and left for 5 min at room temperature. Eight microliters of the designed siRNA primer PIM2-Homo-525 (GenePharma), as well as siRNA GAPDH (positive control) and siRNA NC (negative control), was added to 200 μL of serum-free medium. The mixture was incubated for 20 min at room temperature before mixing with transfection reagents to form transfection complexes. The siRNA transfection reagent mixture was added to the cell culture plate, gently mixed, and incubated in a CO₂ incubator. The proteins were extracted after 72 h of transfection for subsequent experiments.