s4215

Protocol tips

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Seed 1 × 10^5 cells per well	DNMT1 siRNA sequences:5′-GGAUGAGAAGAGACGUAGAtt-3′ and 5′-UCUACGUCUCUUCUCAUCCtg-3′, and the DNMT3B siRNA sequences were 5′-GCUCGUCUCCUAUCGAAAAtt-3′ and 5′-UUUUCGAUAGGAGACGAGCtt-3′. 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 for 2 days at 37°C.

Upstream tips
Seed 1 × 10^5 cells per well
Protocol tips
DNMT1 siRNA sequences:5′-GGAUGAGAAGAGACGUAGAtt-3′ and 5′-UCUACGUCUCUUCUCAUCCtg-3′, and the DNMT3B siRNA sequences were 5′-GCUCGUCUCCUAUCGAAAAtt-3′ and 5′-UUUUCGAUAGGAGACGAGCtt-3′. 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 for 2 days at 37°C.

Publication protocol

The DNMT1 and 3b-specific siRNA duplexes were chosen from the GenBank sequences (accession #NM_001379.1 and #NM_175849.1, respectively) and synthesized by Ambion Inc. (Austin, TX, USA). The DNMT1 siRNA sequences were 5′-GGAUGAGAAGAGACGUAGAtt-3′ and 5′-UCUACGUCUCUUCUCAUCCtg-3′, and the DNMT3B siRNA sequences were 5′-GCUCGUCUCCUAUCGAAAAtt-3′ and 5′-UUUUCGAUAGGAGACGAGCtt-3′. Negative control siRNA was also purchased from Ambion Inc. (Catalog #4390843). Final concentrations were 7 nM. The OptiMEM (Gibco BRL Inc.) and Lipofectamine 2000 Transfection Kits (Invitrogen) were used for siRNA transfections. Cells were plated at 1 × 105 cells per well in culture dishes for overnight growth and transfected by siRNA on the following day. Six hours after transfection, the medium was removed and replaced with DMEM, and the cells were allowed to grow for 48h. Then, viability, apoptosis, cell cycle and gene expression were assayed.

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