Accell Human VDAC1 (7416) siRNA - Set of 4

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 150,000 cells/well	siRNA concentration- 5–100 nmol/l si-hVDAC1- S: 238-5′ACACUAGGCACCGAGAUUA3′-256 and AS: 5′UAAUCUCGGUGCCUAGUGU3′; si-hVDAC1 1/A- S: -238-5′ACACUAGGCACCGAGAUUA 3′- 256, and AS: 5′UAAUCUCGGUGCCUAGUGU3′-256; si-hVDAC1 2/A- S: 238-5′ ACACUAGGCACCGAGAUUA3′-256 and AS: 238-5′UAAUCUCGGUGCCUAGUGU3′; si-hVDAC1 2/B-, S: 238- 5′ACACUAGGCACCGAGA UUA3′-256 and AS: 238-5′UAAUCUCGGUGCCUAGUGU3′-256 Mix the contents (dilute the siRNA and diluted DharmaFECT transfection reagent in serum-free medium) and incubate for 5 minutes at room temperature. Add mixture to cells and incubate for 20 minutes at room temperature. Replace culture medium with transfection medium and incubate cells at 37°C in 5% CO2 for 48 hours.

Upstream tips
Seed 150,000 cells/well
Protocol tips
siRNA concentration- 5–100 nmol/l si-hVDAC1- S: 238-5′ACACUAGGCACCGAGAUUA3′-256 and AS: 5′UAAUCUCGGUGCCUAGUGU3′; si-hVDAC1 1/A- S: -238-5′ACACUAGGCACCGAGAUUA 3′- 256, and AS: 5′UAAUCUCGGUGCCUAGUGU3′-256; si-hVDAC1 2/A- S: 238-5′ ACACUAGGCACCGAGAUUA3′-256 and AS: 238-5′UAAUCUCGGUGCCUAGUGU3′; si-hVDAC1 2/B-, S: 238- 5′ACACUAGGCACCGAGA UUA3′-256 and AS: 238-5′UAAUCUCGGUGCCUAGUGU3′-256 Mix the contents (dilute the siRNA and diluted DharmaFECT transfection reagent in serum-free medium) and incubate for 5 minutes at room temperature. Add mixture to cells and incubate for 20 minutes at room temperature. Replace culture medium with transfection medium and incubate cells at 37°C in 5% CO2 for 48 hours.

Publication protocol

Nonmodified and 2′-O-methyl-modified hVDAC1-siRNAs were synthesized by Dharmacon or were obtained from Genepharma (Suzhou, China). The following sequences (names underlined) were used, with 2′-O-methyl-modified nucleotides indicated in bold and underlined (nucleotide positions are provided for sense (S) and antisense (AS) sequences): si-hVDAC1- S: 238-5′ACACUAGGCACCGAGAUUA3′-256 and AS: 5′UAAUCUCGGUGCCUAGUGU3′; si-hVDAC1 1/A- S: -238-5′ACACUAGGCACCGAGAUUA 3′- 256, and AS: 5′UAAUCUCGGUGCCUAGUGU3′-256; si-hVDAC1 2/A- S: 238-5′ ACACUAGGCACCGAGAUUA3′-256 and AS: 238-5′UAAUCUCGGUGCCUAGUGU3′; si-hVDAC1 2/B-, S: 238- 5′ACACUAGGCACCGAGA UUA3′-256 and AS: 238-5′UAAUCUCGGUGCCUAGUGU3′-256; si-mVDAC1- S: 238-5′ACCCUGGGCACUGAGAUCA3′-256 and AS: 238-5′UGAUCUCAGUGCCCAGGGU 3′-256; and Scrambled- S: -5′GCAAACAUCCCAGAGGUAU3′ and AS : -5′AUACCUCUGGGAUGUUUGC3′.

Cells were seeded (150,000 cells/well) on six-well culture dishes to 30–50% confluence and transfected with 5–100 nmol/l siRNA-hVDAC1 using the JetPRIME reagent in the case of HeLa and A549, PC-3, HepG2, U87, HaCat, and Min-6 and Panc-1cells, while HCT116 and HEK293, cells were transfected using Dharmafect, all according to the manufacturers' instructions.

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