siRNA / miRNA gene silencing Human - HT-1376 CD74

Gene silencing through the use of small interfering RNA (siRNA) has become a primary tool for identifying disease-causing genes. There are several aspects for preparing and delivering effective siRNA to knockdown a target gene. The length of siRNA should be 21–23nt long with G/C content 30–50%. If a validated siRNA sequence for your target gene is not available, use siRNA generated against the entire target gene ORF. Always work with two or three different siRNA constructs to get reliable results. If you are not sure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM and use siRNA-specific transfection reagent to ensure efficient siRNA delivery in a wide range of cells.

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CD74 siRNA and shRNA Plasmids (h)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	HT-1376 (G3) cell lines were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). HT-1376 were all cultured in RPMI-1640 (Hyclone; GE Healthcare Life Sciences) medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were routinely cultured in a 37°C humidified incubator with 5% CO2 and 95% O2. The CD74 shRNA lentiviral particles (cat. no., sc-35023-V; 1.0×106/200 µl; Santa Cruz Biotechnology, Inc.) targeting the human CD74 transcript and control shRNA lentiviral particles (cat. no., sc-108080; 1.0×106/200 µl; Santa Cruz Biotechnology, Inc.) were used to knock down CD74 in HT-1376 cells. Cells were seeded at 2×105 cells/well in 6-well plates (Corning Incorporated, Corning, NY, USA) and grown to 60% confluence on the day of transfection. The media was removed from the plate wells and replaced with 2 ml complete medium containing Polybrene (cat. no., sc-134220; Santa Cruz Biotechnology, Inc.) at a final concentration of 5 µg/ml. Cells were transfected with control (scramble, 20 µl) or CD74 shRNA lentiviral particles (20 µl) diluted in media according to the manufacturer's protocol in 48 h. Infected cells were selected with puromycin (5 µg/ml; cat. no., 11811-023; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). A total of 72 h after transfection, transfected cells were verified by RT-PCR and western blotting analysis, comparing untreated and scramble cells.

Protocol tips
HT-1376 (G3) cell lines were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). HT-1376 were all cultured in RPMI-1640 (Hyclone; GE Healthcare Life Sciences) medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were routinely cultured in a 37°C humidified incubator with 5% CO2 and 95% O2. The CD74 shRNA lentiviral particles (cat. no., sc-35023-V; 1.0×106/200 µl; Santa Cruz Biotechnology, Inc.) targeting the human CD74 transcript and control shRNA lentiviral particles (cat. no., sc-108080; 1.0×106/200 µl; Santa Cruz Biotechnology, Inc.) were used to knock down CD74 in HT-1376 cells. Cells were seeded at 2×105 cells/well in 6-well plates (Corning Incorporated, Corning, NY, USA) and grown to 60% confluence on the day of transfection. The media was removed from the plate wells and replaced with 2 ml complete medium containing Polybrene (cat. no., sc-134220; Santa Cruz Biotechnology, Inc.) at a final concentration of 5 µg/ml. Cells were transfected with control (scramble, 20 µl) or CD74 shRNA lentiviral particles (20 µl) diluted in media according to the manufacturer's protocol in 48 h. Infected cells were selected with puromycin (5 µg/ml; cat. no., 11811-023; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). A total of 72 h after transfection, transfected cells were verified by RT-PCR and western blotting analysis, comparing untreated and scramble cells.

Protocol tips

HT-1376 (G3) cell lines were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). HT-1376 were all cultured in RPMI-1640 (Hyclone; GE Healthcare Life Sciences) medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were routinely cultured in a 37°C humidified incubator with 5% CO2 and 95% O2. The CD74 shRNA lentiviral particles (cat. no., sc-35023-V; 1.0×106/200 µl; Santa Cruz Biotechnology, Inc.) targeting the human CD74 transcript and control shRNA lentiviral particles (cat. no., sc-108080; 1.0×106/200 µl; Santa Cruz Biotechnology, Inc.) were used to knock down CD74 in HT-1376 cells. Cells were seeded at 2×105 cells/well in 6-well plates (Corning Incorporated, Corning, NY, USA) and grown to 60% confluence on the day of transfection. The media was removed from the plate wells and replaced with 2 ml complete medium containing Polybrene (cat. no., sc-134220; Santa Cruz Biotechnology, Inc.) at a final concentration of 5 µg/ml. Cells were transfected with control (scramble, 20 µl) or CD74 shRNA lentiviral particles (20 µl) diluted in media according to the manufacturer's protocol in 48 h. Infected cells were selected with puromycin (5 µg/ml; cat. no., 11811-023; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). A total of 72 h after transfection, transfected cells were verified by RT-PCR and western blotting analysis, comparing untreated and scramble cells.

Manufacturer protocol Publication protocol 1 Relevant paper

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