siRNA / miRNA gene silencing Human - HT-29 B7-H6/NCR3LG1

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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Found 1 matching solution for this experiment

ON-TARGETplus Human NCR3LG1 (374383) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	To target B7H6 expression in tumor cell lines, a mix of four siRNAs complementary to human B7H6 (ON‐TARGETplus SMARTpool, Dharmacon, Thermo Fisher Scientific) was used. In a 24‐well plate, 3.6 × 105 cells were plated in each well. After 24 h, 7.2 pmol siRNA (B7H6 or control) was mixed with 1.2 μL RNAiMAX (Thermo Fisher Scientific) (both dissolved in OptiMEM), incubated at room temperature for 20 min and added to the cells in 500 μL of complete medium. 60–66 h after transfection start, cells were washed twice in OptiMEM, and complete medium was added. Cells were harvested for reporter assays 72 h after transfection start. Surface expression of B7H6 was analyzed by flow cytometry at 72 and 96 h after transfection start.

Protocol tips
To target B7H6 expression in tumor cell lines, a mix of four siRNAs complementary to human B7H6 (ON‐TARGETplus SMARTpool, Dharmacon, Thermo Fisher Scientific) was used. In a 24‐well plate, 3.6 × 105 cells were plated in each well. After 24 h, 7.2 pmol siRNA (B7H6 or control) was mixed with 1.2 μL RNAiMAX (Thermo Fisher Scientific) (both dissolved in OptiMEM), incubated at room temperature for 20 min and added to the cells in 500 μL of complete medium. 60–66 h after transfection start, cells were washed twice in OptiMEM, and complete medium was added. Cells were harvested for reporter assays 72 h after transfection start. Surface expression of B7H6 was analyzed by flow cytometry at 72 and 96 h after transfection start.

Protocol tips

To target B7H6 expression in tumor cell lines, a mix of four siRNAs complementary to human B7H6 (ON‐TARGETplus SMARTpool, Dharmacon, Thermo Fisher Scientific) was used. In a 24‐well plate, 3.6 × 105 cells were plated in each well. After 24 h, 7.2 pmol siRNA (B7H6 or control) was mixed with 1.2 μL RNAiMAX (Thermo Fisher Scientific) (both dissolved in OptiMEM), incubated at room temperature for 20 min and added to the cells in 500 μL of complete medium. 60–66 h after transfection start, cells were washed twice in OptiMEM, and complete medium was added. Cells were harvested for reporter assays 72 h after transfection start. Surface expression of B7H6 was analyzed by flow cytometry at 72 and 96 h after transfection start.

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