s15827

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration-10, 50 and 100 nM. sense strand 5′-GCUCAGCAGGAAAGGUGUUUU-3′ and antisense strand 5′-AACACCUUUCCUGCUGAGCUU-3′ Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio).  Incubate for 5 minutes at room temperature and add to cells.  Incubate cells for 1–3 days at 37°C.

Protocol tips
siRNA concentration-10, 50 and 100 nM. sense strand 5′-GCUCAGCAGGAAAGGUGUUUU-3′ and antisense strand 5′-AACACCUUUCCUGCUGAGCUU-3′ Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio).  Incubate for 5 minutes at room temperature and add to cells.  Incubate cells for 1–3 days at 37°C.

Publication protocol

For CDC7 siRNA treatment, a custom double-stranded siRNA with sequence: sense strand 5′-GCUCAGCAGGAAAGGUGUUUU-3′ and antisense strand 5′-AACACCUUUCCUGCUGAGCUU-3′ (Dharmacon, Thermo Scientific, Lafayette, CO, USA) was used, with non-targeting siRNA (Ambion, Invitrogen, Grand Island, NY, USA) as a negative control. To control for a non-target effect, an alternative CDC7 siRNA was also used with the sequence: sense strand 5′-GCTCAGCAGGAAAGGTGTTTT-3′ and antisense strand 5′-AACACCTTTCCTGCTGAGCTT-3′ (Dharmacon, Thermo Scientific, Lafayette, CO, USA). For the PANC-1 and IMR-90 lines, siRNA was diluted to 10, 50 and 100 nM concentrations in Opti-MEM (Gibco), and complexed with lipofectamine RNAiMAX (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. Due to poor efficacy of standard transfection reagents in the Capan-1 cell line, transfection was carried out via electroporation using the Amaxa Cell Line Nucleofector Kit V (Lonza Cologne AG, Cologne, Germany) with 50 and 100 nM siRNA, according to the manufacturer's instructions [39].

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