siRNA / miRNA gene silencing Human - MDA-MB-468 SNAI 1

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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SNAI 1 siRNA and shRNA Plasmids (h)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream 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.

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.

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.

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