SNAI 1 siRNA and shRNA Plasmids (h)

Protocol tips

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	MDA-MB-468 breast adenocarcinoma cell lines were purchased from the Pasteur Institute, Tehran, Iran, and was maintained in RPMI-1640 medium supplemented with 10% FBS, 1% antibiotics (100 unit/ml penicillin and 100 μg/ml streptomycin). Cell lines were cultured at 37 °C in a humidified atmosphere containing 5% carbon dioxide. Every day we changed the culture medium and passaged the cells when they reached 80% to 90% confluences. Snail1 targeting siRNAs were designed by using Santacruz Biotechnology California, USA (http://www.scbt.com/datasheet-38398-snai-1-sirna-h.html). This designed siRNA sequences were blasted against the human genome database to eliminate cross-silence phenomenon with non-target genes. Scrambled siRNA (Santacruz) that does not target any gene was used as the negative control siRNA. Pooled human snail1 siRNA is a pool of 3 different siRNA duplexes sequences including siRNA duplex A, Sense: GGACUUUGAUGAAGACCAUtt and Antisense: AUGGUCUUCAUCAAAGUCCtt, siRNA duplex B, Sense: CACGAGGUGUGACUAACUAtt and Antisense: UAGUUAGUCACACCUCGUGtt, siRNA duplex C, Sense: GCGAGCUGCAGGACUCUAAtt and Antisense: UUAGAGUCCUGCAGCUCGCtt. We needed to acquire the time and dosage affected by the siRNA. To do it average dosage of 60 was treated in three times of 24, 48 and 72 h to gain the effective time and three dosages of 40, 60 and 80 pmol were evaluated in the gained effective time to acquire the effective dosage. Cells were transfected with siRNA and transfection reagent according to the manufacturer’s instructions. Transfection of siRNA was done in three doses of 40, 60 and 80 pmol. Briefly, cells were seeded in a 6-well-plate at a density of 1 × 106 cells/well with antibiotics-free medium 40 min before the transfection. Six μl of the siRNA concentration was mixed with 6 μl transfection reagent in 200 μl optimal medium and were incubated at room temperature for 30 min to form a complex. After rinsing cells with PBS, 212 μl transfection mixtures were supplemented to each well with 800 μl optimal medium. Six hours after the transfection, the medium was replaced with fresh 1 ml RPMI-1640 medium containing 20% FBS. Overall, 24, 48 and 72 h after the transfection, cells was collected for RNA and protein isolation.

Protocol tips

MDA-MB-468 breast adenocarcinoma cell lines were purchased from the Pasteur Institute, Tehran, Iran, and was maintained in RPMI-1640 medium supplemented with 10% FBS, 1% antibiotics (100 unit/ml penicillin and 100 μg/ml streptomycin). Cell lines were cultured at 37 °C in a humidified atmosphere containing 5% carbon dioxide. Every day we changed the culture medium and passaged the cells when they reached 80% to 90% confluences. Snail1 targeting siRNAs were designed by using Santacruz Biotechnology California, USA (http://www.scbt.com/datasheet-38398-snai-1-sirna-h.html). This designed siRNA sequences were blasted against the human genome database to eliminate cross-silence phenomenon with non-target genes. Scrambled siRNA (Santacruz) that does not target any gene was used as the negative control siRNA. Pooled human snail1 siRNA is a pool of 3 different siRNA duplexes sequences including siRNA duplex A, Sense: GGACUUUGAUGAAGACCAUtt and Antisense: AUGGUCUUCAUCAAAGUCCtt, siRNA duplex B, Sense: CACGAGGUGUGACUAACUAtt and Antisense: UAGUUAGUCACACCUCGUGtt, siRNA duplex C, Sense: GCGAGCUGCAGGACUCUAAtt and Antisense: UUAGAGUCCUGCAGCUCGCtt. We needed to acquire the time and dosage affected by the siRNA. To do it average dosage of 60 was treated in three times of 24, 48 and 72 h to gain the effective time and three dosages of 40, 60 and 80 pmol were evaluated in the gained effective time to acquire the effective dosage. Cells were transfected with siRNA and transfection reagent according to the manufacturer’s instructions. Transfection of siRNA was done in three doses of 40, 60 and 80 pmol. Briefly, cells were seeded in a 6-well-plate at a density of 1 × 106 cells/well with antibiotics-free medium 40 min before the transfection. Six μl of the siRNA concentration was mixed with 6 μl transfection reagent in 200 μl optimal medium and were incubated at room temperature for 30 min to form a complex. After rinsing cells with PBS, 212 μl transfection mixtures were supplemented to each well with 800 μl optimal medium. Six hours after the transfection, the medium was replaced with fresh 1 ml RPMI-1640 medium containing 20% FBS. Overall, 24, 48 and 72 h after the transfection, cells was collected for RNA and protein isolation.

Publication protocol

MDA-MB-468 breast adenocarcinoma cell lines were purchased from the Pasteur Institute, Tehran, Iran, and was maintained in RPMI-1640 medium supplemented with 10% FBS, 1% antibiotics (100 unit/ml penicillin and 100 μg/ml streptomycin). Cell lines were cultured at 37 °C in a humidified atmosphere containing 5% carbon dioxide. Every day we changed the culture medium and passaged the cells when they reached 80% to 90% confluences. Snail1 targeting siRNAs were designed by using Santacruz Biotechnology California, USA (http://www.scbt.com/datasheet-38398-snai-1-sirna-h.html). This designed siRNA sequences were blasted against the human genome database to eliminate cross-silence phenomenon with non-target genes. Scrambled siRNA (Santacruz) that does not target any gene was used as the negative control siRNA. Pooled human snail1 siRNA is a pool of 3 different siRNA duplexes sequences including siRNA duplex A, Sense: GGACUUUGAUGAAGACCAUtt and Antisense: AUGGUCUUCAUCAAAGUCCtt, siRNA duplex B, Sense: CACGAGGUGUGACUAACUAtt and Antisense: UAGUUAGUCACACCUCGUGtt, siRNA duplex C, Sense: GCGAGCUGCAGGACUCUAAtt and Antisense: UUAGAGUCCUGCAGCUCGCtt. We needed to acquire the time and dosage affected by the siRNA. To do it average dosage of 60 was treated in three times of 24, 48 and 72 h to gain the effective time and three dosages of 40, 60 and 80 pmol were evaluated in the gained effective time to acquire the effective dosage. Cells were transfected with siRNA and transfection reagent according to the manufacturer’s instructions. Transfection of siRNA was done in three doses of 40, 60 and 80 pmol. Briefly, cells were seeded in a 6-well-plate at a density of 1 × 106 cells/well with antibiotics-free medium 40 min before the transfection. Six μl of the siRNA concentration was mixed with 6 μl transfection reagent in 200 μl optimal medium and were incubated at room temperature for 30 min to form a complex. After rinsing cells with PBS, 212 μl transfection mixtures were supplemented to each well with 800 μl optimal medium. Six hours after the transfection, the medium was replaced with fresh 1 ml RPMI-1640 medium containing 20% FBS. Overall, 24, 48 and 72 h after the transfection, cells was collected for RNA and protein isolation.

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