INTERFERin®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration- 20 nmol/L siRNA SEQUENCE- GGAGAUUGAUGGUCUACUA-3′ and 5′-AACAGCCAUGGAUACACUUGATT-3′ Add INTERFERin® to siRNA duplexes and incubate for 10min at RT after homogenization. Remove culture medium from cells and add incubate mixture. Incubate the plate at 37°C for 24 to 72 h.

Protocol tips
siRNA concentration- 20 nmol/L siRNA SEQUENCE- GGAGAUUGAUGGUCUACUA-3′ and 5′-AACAGCCAUGGAUACACUUGATT-3′ Add INTERFERin® to siRNA duplexes and incubate for 10min at RT after homogenization. Remove culture medium from cells and add incubate mixture. Incubate the plate at 37°C for 24 to 72 h.

Publication protocol

Briefly, a single pulse of 20 nmol/L of small interfering RNA (siRNA) was administered to the cells at 50–70% confluency via transfection with 8 μL INTERFERin (Polyplus) in Opti-MEM for the time indicated in the figure legends. FANCA siRNAs were Smartpools (Dharmacon) and from Invitrogen (FANCA#2) 5′-GGGUCAAGAGGGAAAAAUATT-3′. MiTF siRNAs were from Invitrogen: 5′-GGUGAAUCGGAUCAUCAAG-3′ (siMiTF1) or 5′-AGCAGUACCUUUCUACCACTT-3′(siMiTF2). Human FANCD2 was targeted with two individual siRNA from Invitrogen 5′-GGAGAUUGAUGGUCUACUA-3′ and 5′-AACAGCCAUGGAUACACUUGATT-3′. Mouse Fancd2 was targeted with the Smartpool siRNA from Dharmacon. In the case of double transfection, the same total amount of siRNA was used with a control siRNA added to the single siRNA of interest.

pCDNA3 (empty vector), pFLAG-FANCD2 and pFLAG-FANCA were a gift of A. Constantinou’s lab (Montpellier). Briefly, 1 μg of plasmid was transfected to the cells using 6 μL JetPEI (Polyplus) in 150 mM NaCl for the time indicated in the figure legends. The medium was changed 6 h after transfection. For siRNA and plasmid cotransfection, the siRNA were transfected 8 h after seeding; the plasmids were transfected 16 h later.

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Manufacturer protocol

Download the product protocol from Polyplus transfections for INTERFERin® below.

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