siRNA transfection

For siRNA transfection, pancreatic cancer cells were seeded in T-25 flasks (4–7 × 10⁵ cells) and incubated 72h in 37°C. The cells were then washed twice with phosphate buffered saline (PBS) and received growth medium without FBS, together with lipofectamine 2000 and negative control or anti-RBM3 (s11858 + s11860) siRNA in OptiMEM to a final siRNA concentration of 50 nM. The transfection was stopped after 4.5h, medium changed to full growth medium and the cells were left to recover overnight. The following day, cells were harvested and spun down to pellets. The pellets were either fixated, dehydrated and embedded in paraffin for immunohistochemistry or resuspended in RLT buffer (QIAGEN) and stored in -20°C for qPCR.