ON-TARGETplus Mouse Nr1h3 (22259) siRNA - SMARTpool

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	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.

Publication protocol

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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