siRNA / miRNA gene silencing Mouse - M210B4 LXR‐α/Nr1h3

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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ON-TARGETplus Mouse Nr1h3 (22259) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Both LXR‐α and LXR‐β siRNAs (ON‐TARGETplus SMARTpool Catalog No. L‐040649‐01‐0010 and L‐042839‐00‐0010) were obtained from Dharmacon (Lafayette, CO, USA). To knock down LXRs, M2 cells at 70% confluence in 6 well plates were transfected with siRNA using DharmaFECT transfection reagent (Dharmacon) to a final concentration of 25 nM of each siRNA.42 Knockdown of target genes was monitored at the mRNA level by quantitative real‐time PCR. At 100% confluence, transfected cells were treated with control vehicle or 5 µM 20S . After a 2 day incubation, HES‐1 and HEY‐1 mRNA expression was measured by quantitative real‐time PCR. Both HES‐1 and HEY‐1 siRNAs were obtained from QIAGEN (Valencia, CA, USA). To knock down HES‐1 or HEY‐1, M2 cells at 70% confluence in 6 well plates were transfected with siRNA using DharmaFECT transfection reagent (Dharmacon) to a final concentration of 50 nM of each siRNA. At 100% confluence, transfected cells were treated with 5 µM 20S . After 3 days of incubation, alkaline phosphatase (ALP ), bone sialoprotein (BSP ), and osteocalcin (OCN ) mRNA expression was measured by quantitative real‐time PCR.

Protocol tips
Both LXR‐α and LXR‐β siRNAs (ON‐TARGETplus SMARTpool Catalog No. L‐040649‐01‐0010 and L‐042839‐00‐0010) were obtained from Dharmacon (Lafayette, CO, USA). To knock down LXRs, M2 cells at 70% confluence in 6 well plates were transfected with siRNA using DharmaFECT transfection reagent (Dharmacon) to a final concentration of 25 nM of each siRNA.42 Knockdown of target genes was monitored at the mRNA level by quantitative real‐time PCR. At 100% confluence, transfected cells were treated with control vehicle or 5 µM 20S . After a 2 day incubation, HES‐1 and HEY‐1 mRNA expression was measured by quantitative real‐time PCR. Both HES‐1 and HEY‐1 siRNAs were obtained from QIAGEN (Valencia, CA, USA). To knock down HES‐1 or HEY‐1, M2 cells at 70% confluence in 6 well plates were transfected with siRNA using DharmaFECT transfection reagent (Dharmacon) to a final concentration of 50 nM of each siRNA. At 100% confluence, transfected cells were treated with 5 µM 20S . After 3 days of incubation, alkaline phosphatase (ALP ), bone sialoprotein (BSP ), and osteocalcin (OCN ) mRNA expression was measured by quantitative real‐time PCR.

Protocol tips

Both LXR‐α and LXR‐β siRNAs (ON‐TARGETplus SMARTpool Catalog No. L‐040649‐01‐0010 and L‐042839‐00‐0010) were obtained from Dharmacon (Lafayette, CO, USA). To knock down LXRs, M2 cells at 70% confluence in 6 well plates were transfected with siRNA using DharmaFECT transfection reagent (Dharmacon) to a final concentration of 25 nM of each siRNA.42 Knockdown of target genes was monitored at the mRNA level by quantitative real‐time PCR. At 100% confluence, transfected cells were treated with control vehicle or 5 µM 20S . After a 2 day incubation, HES‐1 and HEY‐1 mRNA expression was measured by quantitative real‐time PCR. Both HES‐1 and HEY‐1 siRNAs were obtained from QIAGEN (Valencia, CA, USA). To knock down HES‐1 or HEY‐1, M2 cells at 70% confluence in 6 well plates were transfected with siRNA using DharmaFECT transfection reagent (Dharmacon) to a final concentration of 50 nM of each siRNA. At 100% confluence, transfected cells were treated with 5 µM 20S . After 3 days of incubation, alkaline phosphatase (ALP ), bone sialoprotein (BSP ), and osteocalcin (OCN ) mRNA expression was measured by quantitative real‐time PCR.

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