Smartpool ON-TARGETplus siRNAs targeting CTNNB1 and WLS were obtained from Dharmacon. The ON-TARGETplus nontargeting siRNA #2 was used as negative control. Cells were reverse-transfected in a 96-well plate using a total of 0.2 μl DharmaFECT formulation 4 (Thermo Fischer Scientific) and 25 nM of each siRNA per well. Following 72 hours of incubation, the effect on knockdown was determined by Western blotting.
Dharmafect formulation 4
Manufactured by Thermo Fisher Scientific
DharmaFECT formulation 4 is a transfection reagent for the delivery of nucleic acids into mammalian cells. It is designed to effectively transfect a wide range of cell types with high efficiency.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using dharmafect formulation 4
1
Optimized Silencing of CTNNB1 and WLS
2
Targeted siRNA Knockdown Across Wnt Pathway
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SiRNA-mediated gene knockdown was performed as reported previously14 (link),50 (link). Smartpool ON-TARGETplus siRNAs targeting AXIN1, AXIN2 or APC were purchased from Dharmacon (Supplemental Table S4 ). The ON-TARGETplus Non-targeting siRNA #2 was used as negative control. Cells were reverse-transfected in a 24-well plate using a total of 0.8 µl DharmaFECT formulation 4 (Thermo-Fisher Scientific) and 25 nM of each siRNA per well. Following 72 h incubation, the effect of knock-down was tested by qRT-PCR. Alternatively, for combined siRNA/β-catenin reporter assays, the siRNAs were co-transfected with WRE or MRE vectors and CMV-Renilla using lipofectamin 2000, and measured after 48 h.
3
siRNA-mediated Knockdown and β-catenin Assay
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Smartpool ON-TARGETplus siRNAs targeting STRAP were purchased from Dharmacon. The ON-TARGETplus Non-targeting siRNA #2 was used as negative control. Cells were reversetransfected in a 24-well plate using a total of 0.8 µl DharmaFECT formulation 4 (Thermo Fischer Scientific) and 25nM of each siRNA per well. Following 72 h incubation, the effect of knockdown was tested by western blotting. Alternatively, 48h after reverse transfection, the cells were transfected with WRE or MRE vectors and CMV-Renilla for β-catenin reporter assay.
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