SnoA/N siRNA (h)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 1×105 cells/well in 24-well plate	siRNA concentration 0 nmol/L, 50 nmol/L, 75 nmol/L, and 100 nmol/L, Add diluted siRNA to diluted Lipofectamine Reagent   Incubate for 5 min at RT and add to cells.  Replace medium after 6h and re-incubate cells at 37°C.

Upstream tips
Seed 1×105 cells/well in 24-well plate
Protocol tips
siRNA concentration 0 nmol/L, 50 nmol/L, 75 nmol/L, and 100 nmol/L, Add diluted siRNA to diluted Lipofectamine Reagent   Incubate for 5 min at RT and add to cells.  Replace medium after 6h and re-incubate cells at 37°C.

Publication protocol

SnoN siRNAs, labelled by florescence FAM (FAM-siRNAs), and negative control siRNAs were synthesised by Shanghai Jima Biotechnology Co., Ltd. (Shanghai, China). For the SnoN siRNA-A, the sense strand was 5’-GGGCUUUGAAUCAGCUAAATT-3’ and the antisense strand was 5’-UUUAGCUGAUUCAAAGCCCTT-3’. For the SnoN siRNA-B, the sense strand was 5’-GGCCCAGUUAAAGGAAACUTT-3’ and the antisense strand was 5’-AGUUUCCUUUAACUGGGCCTT-3’. For the SnoN siRNA-C, the sense strand was 5’-GAGGCAAGUAAGUCCAUAUTT-4’ and the antisense strand was 5’-AUAUGGACUUACUUGCCUCTT-3’. For the negative control siRNA, the sense strand was 5’-UUCUCCGAACGUGUCACGUTT-3’ and the antisense strand was 5’-ACGUGACACGUUCGGAGAATT-3’. The SnoN primers were forward, 5’-AGAGACTCTGTTTGCCCCAAGT-3’ and reverse, 5’-CATGCTAAACTTCTCCTTCATTTC-3’. The β-actin primers were forward, 5’-TTCTGTGGCATCCACGAAACT-3’ and reverse, 5’-GAAGCATTTGCGGTGGACGAT-3’.

siRNA transfection
The pancreatic cancer cells were seeded at 1×105 cells/well in 24-well plates 1 day before transfection. Medium without antibiotics was added to each well so that the cells grew to 50-70% confluence, when the transfection was conducted. The siRNA-Lipo mixture was prepared according to the manufacturer's instructions. To test the transfection efficiency of the FAM-siRNAs-Lipo mixture at different concentrations, 0, 1, 1.5 and 2 μl of Lipo were diluted with 50 μl Opti-MEM, and 0, 10, 15 and 20 μl FAM-siRNAs, respectively, were added at 50 μl/well and mixed. The 100 μl mixture was added to 300 μl of Opti-MEM including the pancreatic cancer cells. The concentrations of the four groups were 0 nmol/L, 50 nmol/L, 75 nmol/L, and 100 nmol/L, respectively. Six hours later, the medium was replaced with DMEM supplemented with 10% FBS. The expression of FAM-siRNAs was analysed with a flow cytometer.

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Manufacturer protocol

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