ON-TARGETplus Human CYP2B6 (1555) siRNA - SMARTpool

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Using siRNA to suppress CYP proteins was conducted by modification of the method published by Vozza-Brown et al. [37]. After 5 h incubation with hepatocyte plating medium, cells were washed and treated with Dharmacon ON-TARGETplus human CYP3A4 or CYP2B6 SMARTpool siRNA. Positive ON-TARGETplus GAPD human control pool and negative control ON-TARGETplus non-targeting pool were also included (Table 2). A non-siRNA treated control was also included to differentiate between the loss of gene expression due to endogenous degradation and loss due to siRNA treatment. 1.25 μL of transfection reagent Lipofectamine RNAiMAX™ was complexed with 50 or 100 nM ON-TARGETplus CYP3A4 or CYP2B6 siRNA, respectively, in reduced serum opti-MEM® I medium for 30 min prior to addition to cell culture. Hepatocytes were exposed to siRNA overnight for 15 h before the cells were washed and replaced with standard hepatocyte maintenance medium. Cells were subsequently incubated at 37 °C with 5% (v/v) CO2 for 48, 72 and 96 h post CYP3A4 siRNA treatment, and for 12, 24, 36, 48 and 60 h after CYP2B6 siRNA treatment. CYP3A4 and CYP2B6 metabolic activity, mRNA and protein expression were assessed at these time points. Time 0 h was taken after 5 h incubation with plating media and prior to siRNA transfection and maintenance media replacement. Following siRNA transfection, maintenance media was replaced every 24 h.

Protocol tips

Using siRNA to suppress CYP proteins was conducted by modification of the method published by Vozza-Brown et al. [37]. After 5 h incubation with hepatocyte plating medium, cells were washed and treated with Dharmacon ON-TARGETplus human CYP3A4 or CYP2B6 SMARTpool siRNA. Positive ON-TARGETplus GAPD human control pool and negative control ON-TARGETplus non-targeting pool were also included (Table 2). A non-siRNA treated control was also included to differentiate between the loss of gene expression due to endogenous degradation and loss due to siRNA treatment. 1.25 μL of transfection reagent Lipofectamine RNAiMAX™ was complexed with 50 or 100 nM ON-TARGETplus CYP3A4 or CYP2B6 siRNA, respectively, in reduced serum opti-MEM® I medium for 30 min prior to addition to cell culture. Hepatocytes were exposed to siRNA overnight for 15 h before the cells were washed and replaced with standard hepatocyte maintenance medium. Cells were subsequently incubated at 37 °C with 5% (v/v) CO2 for 48, 72 and 96 h post CYP3A4 siRNA treatment, and for 12, 24, 36, 48 and 60 h after CYP2B6 siRNA treatment. CYP3A4 and CYP2B6 metabolic activity, mRNA and protein expression were assessed at these time points. Time 0 h was taken after 5 h incubation with plating media and prior to siRNA transfection and maintenance media replacement. Following siRNA transfection, maintenance media was replaced every 24 h.

Publication protocol

Using siRNA to suppress CYP proteins was conducted by modification of the method published by Vozza-Brown et al. [37]. After 5 h incubation with hepatocyte plating medium, cells were washed and treated with Dharmacon ON-TARGETplus human CYP3A4 or CYP2B6 SMARTpool siRNA. Positive ON-TARGETplus GAPD human control pool and negative control ON-TARGETplus non-targeting pool were also included (Table 2). A non-siRNA treated control was also included to differentiate between the loss of gene expression due to endogenous degradation and loss due to siRNA treatment. 1.25 μL of transfection reagent Lipofectamine RNAiMAX™ was complexed with 50 or 100 nM ON-TARGETplus CYP3A4 or CYP2B6 siRNA, respectively, in reduced serum opti-MEM® I medium for 30 min prior to addition to cell culture. Hepatocytes were exposed to siRNA overnight for 15 h before the cells were washed and replaced with standard hepatocyte maintenance medium. Cells were subsequently incubated at 37 °C with 5% (v/v) CO2 for 48, 72 and 96 h post CYP3A4 siRNA treatment, and for 12, 24, 36, 48 and 60 h after CYP2B6 siRNA treatment. CYP3A4 and CYP2B6 metabolic activity, mRNA and protein expression were assessed at these time points. Time 0 h was taken after 5 h incubation with plating media and prior to siRNA transfection and maintenance media replacement. Following siRNA transfection, maintenance media was replaced every 24 h.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing siRNA / miRNA gene silencing Human - Primary Human Hepatocytes CYP2B6 using ON-TARGETplus Human CYP2B6 (1555) siRNA - SMARTpool from Horizon Discovery Ltd..

Manufacturer protocol

Download the product protocol from Horizon Discovery Ltd. for ON-TARGETplus Human CYP2B6 (1555) siRNA - SMARTpool below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing siRNA / miRNA gene silencing Human - Primary Human Hepatocytes CYP2B6 using ON-TARGETplus Human CYP2B6 (1555) siRNA - SMARTpool from Horizon Discovery Ltd.. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.