siRNA / miRNA gene silencing Human - HT-1376 ROCK2

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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Rock-2 siRNA and shRNA Plasmids (h)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Human bladder cancer cell line]HT-1376 (American Type Culture Collection; ATCC) were used in the present study to perform all in vitro cell experiments. Cells were cultivated in Eagle's minimum essential medium containing 10% FBS (both ATCC) at 37°C in a humidified incubator with 5% CO2.ROCK2 small interfering (si)RNA (human; cat. no. sc-29474) and negative control (NC) siRNA (cat. no. sc-37007) were purchased from Santa Cruz Biotechnology, Inc. Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to perform all cell transfections with 50 nM siRNA and using aforementioned cell lines at a density of 1×106 cells in 2 ml cell suspension. Treatment with Lipofectamine® 2000 reagent alone was used as the C group and transfection with empty vectors or NC siRNA was used as the NC group. Cells were used for subsequent experiments when LINC01638 and ROCK2 overexpression rates were >200% and ROCK2 knockdown rate was <50%, only. The time interval between transfection and subsequent experimentation was 24 h.

Protocol tips
Human bladder cancer cell line]HT-1376 (American Type Culture Collection; ATCC) were used in the present study to perform all in vitro cell experiments. Cells were cultivated in Eagle's minimum essential medium containing 10% FBS (both ATCC) at 37°C in a humidified incubator with 5% CO2.ROCK2 small interfering (si)RNA (human; cat. no. sc-29474) and negative control (NC) siRNA (cat. no. sc-37007) were purchased from Santa Cruz Biotechnology, Inc. Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to perform all cell transfections with 50 nM siRNA and using aforementioned cell lines at a density of 1×106 cells in 2 ml cell suspension. Treatment with Lipofectamine® 2000 reagent alone was used as the C group and transfection with empty vectors or NC siRNA was used as the NC group. Cells were used for subsequent experiments when LINC01638 and ROCK2 overexpression rates were >200% and ROCK2 knockdown rate was <50%, only. The time interval between transfection and subsequent experimentation was 24 h.

Protocol tips

Human bladder cancer cell line]HT-1376 (American Type Culture Collection; ATCC) were used in the present study to perform all in vitro cell experiments. Cells were cultivated in Eagle's minimum essential medium containing 10% FBS (both ATCC) at 37°C in a humidified incubator with 5% CO2.ROCK2 small interfering (si)RNA (human; cat. no. sc-29474) and negative control (NC) siRNA (cat. no. sc-37007) were purchased from Santa Cruz Biotechnology, Inc. Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to perform all cell transfections with 50 nM siRNA and using aforementioned cell lines at a density of 1×106 cells in 2 ml cell suspension. Treatment with Lipofectamine® 2000 reagent alone was used as the C group and transfection with empty vectors or NC siRNA was used as the NC group. Cells were used for subsequent experiments when LINC01638 and ROCK2 overexpression rates were >200% and ROCK2 knockdown rate was <50%, only. The time interval between transfection and subsequent experimentation was 24 h.

Manufacturer protocol Publication protocol 1 Relevant paper

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