siRNA / miRNA gene silencing Human - MDA-MB-231 AHR

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 Human AHR (196) siRNA - Individual

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	The siRNA knockdowns were performed as detailed in our prior reports [33, 34]. Briefly, 200,000 cells (MCF-7 or MDA-MB-231) in 1 mL of DMEM supplemented with 10% FBS were mixed directly with 100 nM of siRNA that was non-targeting, AHR-targeting or LAT1-targeting and 3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and immediately plated in 35 mm tissue culture plates for 48 hrs. MCF-7 cells were then treated with vehicle (DMSO) or 10 nM TCDD for 16 hrs. Treatments were removed and cells were rinsed once with PBS. For Western blotting, total protein was extracted by scraping cells in 2× Laemmli Sample Buffer containing β-mercaptoethanol (BME). Laemmli sample buffer and BME were purchased from BIO-RAD. Standard Western blotting techniques were used to analyze ∼ 10 μg of protein per sample (please refer to our prior reports for technical details [33, 34]). Western blot analysis of GAPDH was used to confirm equal protein loading. Blots were probed with anti-GAPDH antibody (diluted 1:10,000), anti-AHR antibody (diluted 1:5,000) or anti-LAT1 antibody (diluted 1:2,000) overnight at 4oC, followed by incubation with anti-HRP secondary antibody (1:5000) for 1 hr at room temperature. The blots were then rinsed with PBS + 0.1% tween 20, and then developed with enhanced chemiluminescent substrate Millipore Corp., (Billerica, MA). The anti-GAPDH antibody was purchased from Millipore (Cat #MAB374). The anti-AHR antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, Cat #H-211) and the anti- LAT1 antibody was purchased from Cell Signaling Technology (Danvers, MA, Cat #5347). Densitometry was calculated with ImageJ PC-based software (National Institute of Health). The Student-Newman–Keuls (SNK) post-hoc test was used to determine statistically significant differences among groups following one-way analysis of variance (ANOVA).

Protocol tips
The siRNA knockdowns were performed as detailed in our prior reports [33, 34]. Briefly, 200,000 cells (MCF-7 or MDA-MB-231) in 1 mL of DMEM supplemented with 10% FBS were mixed directly with 100 nM of siRNA that was non-targeting, AHR-targeting or LAT1-targeting and 3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and immediately plated in 35 mm tissue culture plates for 48 hrs. MCF-7 cells were then treated with vehicle (DMSO) or 10 nM TCDD for 16 hrs. Treatments were removed and cells were rinsed once with PBS. For Western blotting, total protein was extracted by scraping cells in 2× Laemmli Sample Buffer containing β-mercaptoethanol (BME). Laemmli sample buffer and BME were purchased from BIO-RAD. Standard Western blotting techniques were used to analyze ∼ 10 μg of protein per sample (please refer to our prior reports for technical details [33, 34]). Western blot analysis of GAPDH was used to confirm equal protein loading. Blots were probed with anti-GAPDH antibody (diluted 1:10,000), anti-AHR antibody (diluted 1:5,000) or anti-LAT1 antibody (diluted 1:2,000) overnight at 4oC, followed by incubation with anti-HRP secondary antibody (1:5000) for 1 hr at room temperature. The blots were then rinsed with PBS + 0.1% tween 20, and then developed with enhanced chemiluminescent substrate Millipore Corp., (Billerica, MA). The anti-GAPDH antibody was purchased from Millipore (Cat #MAB374). The anti-AHR antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, Cat #H-211) and the anti- LAT1 antibody was purchased from Cell Signaling Technology (Danvers, MA, Cat #5347). Densitometry was calculated with ImageJ PC-based software (National Institute of Health). The Student-Newman–Keuls (SNK) post-hoc test was used to determine statistically significant differences among groups following one-way analysis of variance (ANOVA).

Protocol tips

The siRNA knockdowns were performed as detailed in our prior reports [33, 34]. Briefly, 200,000 cells (MCF-7 or MDA-MB-231) in 1 mL of DMEM supplemented with 10% FBS were mixed directly with 100 nM of siRNA that was non-targeting, AHR-targeting or LAT1-targeting and 3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) and immediately plated in 35 mm tissue culture plates for 48 hrs. MCF-7 cells were then treated with vehicle (DMSO) or 10 nM TCDD for 16 hrs. Treatments were removed and cells were rinsed once with PBS. For Western blotting, total protein was extracted by scraping cells in 2× Laemmli Sample Buffer containing β-mercaptoethanol (BME). Laemmli sample buffer and BME were purchased from BIO-RAD. Standard Western blotting techniques were used to analyze ∼ 10 μg of protein per sample (please refer to our prior reports for technical details [33, 34]). Western blot analysis of GAPDH was used to confirm equal protein loading. Blots were probed with anti-GAPDH antibody (diluted 1:10,000), anti-AHR antibody (diluted 1:5,000) or anti-LAT1 antibody (diluted 1:2,000) overnight at 4oC, followed by incubation with anti-HRP secondary antibody (1:5000) for 1 hr at room temperature. The blots were then rinsed with PBS + 0.1% tween 20, and then developed with enhanced chemiluminescent substrate Millipore Corp., (Billerica, MA). The anti-GAPDH antibody was purchased from Millipore (Cat #MAB374). The anti-AHR antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, Cat #H-211) and the anti- LAT1 antibody was purchased from Cell Signaling Technology (Danvers, MA, Cat #5347). Densitometry was calculated with ImageJ PC-based software (National Institute of Health). The Student-Newman–Keuls (SNK) post-hoc test was used to determine statistically significant differences among groups following one-way analysis of variance (ANOVA).

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