Sirna reagent

Manufactured by Santa Cruz Biotechnology

Sourced in United States

SiRNA reagents are a set of laboratory tools used to temporarily inhibit the expression of target genes. They function by introducing small interfering RNA (siRNA) molecules into cells, which can bind to and degrade specific mRNA transcripts, thereby reducing the production of the corresponding proteins.

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Lab products found in correlation

Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Dharmafect transfection reagent, by Thermo Fisher Scientific (1 mentions) Ti e wildfield inverted microscope, by Nikon (1 mentions) Bromophenol blue, by Bio-Rad (1 mentions) Aspirin, by Merck Group (1 mentions) Coomassie brilliant blue g 250, by Bio-Rad (1 mentions) Tris base, by Bio-Rad (1 mentions) Tetramethylethylenediamine, by Bio-Rad (1 mentions) Gapdh mouse monoclonal antibody, by Beyotime (1 mentions) Human ox ldl bt 910, by Thermo Fisher Scientific (1 mentions) Itraq kit, by Thermo Fisher Scientific (1 mentions) Sequence grade modified trypsin, by Promega (1 mentions) 2 d quant kit, by GE Healthcare (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) High performance liquid chromatography system, by Shimadzu (1 mentions) Gadd45a, by Cell Signaling Technology (1 mentions) Cell counting kit 8 cck 8, by MedChemExpress (1 mentions) Dulbecco s modification of eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)

7 protocols using sirna reagent

siRNA Transfection Protocol for Cell Culture

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After the cell density reached 60–80%, siRNA reagent (Santa Cruz Biotechnology, TX, USA) per 3 µL and Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) per 7 µL were diluted and mixed with 100 µL DMEM, respectively, and allowed to stand at room temperature for 30 min. After washing the cells twice with DMEM, 800 µL of siRNA and Lipofectamine 2000 mixture were added to each well, and the cells were incubated for 7 h. Then, 1 mL of medium containing double serum and penicillin–streptomycin solution was added, the mixture was incubated for 24 h and then replaced with conventional medium.

Zeng Q., Zhou T.T., Huang W.J., Huang X.T., Huang L., Zhang X.H., Sang X.X., Luo Y.Y., Tian Y.M., Wu B., Liu L., Luo Z.Q., He B., Liu W, & Tang S.Y. (2023). Asarinin attenuates bleomycin-induced pulmonary fibrosis by activating PPARγ. Scientific Reports, 13, 14706.

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Colorectal Cell Migration Assay

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MX1 was knocked down in two colorectal cell lines, DLD1 and SW450, using commercially available siRNA reagent (sc45260, Santa Cruz Biotechnology, CA) with DharmaFECT transfection reagent (Thermo Fisher) following manufacturer's instructions. The wound-healing assays were performed as previously described (18). For each transfection reaction, including controls mock-transfected with reagent only, multiple replicate wells of a 96-well tissue culture plate were seeded with approximately 5000 cells and incubated for 3 hours to allow the cells to attach. Wounds were created after 24h by manually scratching each well with a yellow pipette tip and the plate was then gently washed with pre-warmed medium to remove detached cells and imaged on a Nikon Ti – E wild field inverted microscope using scan large image option at 10x magnification. The plate was then incubated for 24 hours and imaged again. The images were processed by the NIS Elements software, to calculate wound closure rate (LUNDEBERG et al.,1992) and determine statistical significance whenever judged necessary. After being imaged the cells were lysed, separated by SDS-PAGE and transferred to PDVF membrane. Anti- MX1/2/3 (C1) mouse monoclonal antibody (Santa Cruz Biotechnology, sc-166412) was used for detection of MX1. Imaging was done using Li-Cor infrared Odyssey system.

Croner R.S., Stürzl M., Rau T., Metodieva G., Geppert C.I., Naschberger E., Lausen B, & Metodiev M.V. (2014). Quantitative proteome profiling of lymph node positive vs. negative colorectal carcinomas pinpoints MX1 as a marker for lymph node metastasis. International journal of cancer. Journal international du cancer, 135(12), 2878-2886.

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HSYA Isolation and Analysis in C. tinctorius

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HSYA was isolated and purified from the aqueous extract of C. tinctorius L. by macroporous resin-gel column chromatography, as described previously^{14 (link)}. The molecular weight of HSYA is 612. HSYA was analysed using a high-performance liquid chromatography system (Shimadzu, Kyoto, Japan). iTRAQ kits were purchased from Applied Biosystems (Waltham, MA, USA), the 2-D Quant Kit from GE Healthcare (Pittsburgh, PA, USA), sequence-grade modified trypsin from Promega (Madison, WI, USA), and human ox-LDL (BT-910) from Alfa Aesar (Heysham, Lancashire, UK). Aspirin was the product of Sigma-Aldrich (St. Louis, MO, USA). Bromophenol blue, tetramethylethylenediamine, Coomassie brilliant blue G-250, Bis, low-molecular weight marker, nitrocellulose membrane, Tris-base, and the Bradford method protein assay kit were purchased from Bio-Rad (Hercules, CA, USA). VDAC-2 polyclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA), and GAPDH mouse monoclonal antibody was from Beyotime Biotechnology (Jiangsu, China). Other polyclonal and monoclonal antibodies and siRNA reagents were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), with the secondary antibodies obtained from LI-COR Biosciences (Lincoln, NE, USA). NO and MDA assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Ye F., Wang J., Meng W., Qian J, & Jin M. (2017). Proteomic investigation of effects of hydroxysafflor yellow A in oxidized low-density lipoprotein-induced endothelial injury. Scientific Reports, 7, 17981.

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Chemo-Sensitization Assay Protocol

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COS oligomers (DP 2-7) and glucosamine were purchased from Qingdao BZ Oligo Biotech Co., Ltd., (Qingdao, China), while the cell counting kit-8 (CCK-8) was procured from MedChemExpress, Inc. (Monmouth Junction, NJ, USA). The doxorubicin hydrochloride and Coomassie protein assay reagents were acquired from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Dulbecco’s modification of Eagle’s medium (DMEM), the Trizol reagent, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), RNA-related kits, and the Lipofectamine RNAiMAX Transfection Reagent, as well as the radioimmunoprecipitation assay (RIPA) lysis buffer, were all obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The siRNA reagents were sourced from Santa Cruz Biotechnology (Santa Cruz, CA USA). The antibodies for early growth response protein 1 (EGR1) (catalogue number: 4154S), growth arrest, and the DNA damage-inducible protein alpha (GADD45A) (catalogue number: 4632S), and β-actin (catalogue number: 4970S) were procured from Cell Signaling Technology, Inc. (Danvers, MA, USA). All primers were synthesized by GENEWIZ Biotech Co., Ltd. (Suzhou, China).

Li H., Ji K., Liu P., Geng Y., Gong J., Zhang C., Ding Z., Xu Z, & Shi J. (2023). Chitotriose Enhanced Antitumor Activity of Doxorubicin through Egr1 Upregulation in MDA-MB-231 Cells. Marine Drugs, 22(1), 26.

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Stable Transfection of A549 Cells

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Human type II alveolar epithelial cells (A549) were purchased from and maintained as instructed by the Korean Cell Line Bank (Seoul, Korea). Human A549 cells were stably transfected with pcDNA3 or pcDNA3-BI-1 plasmids using Superfect transfection reagent (QIAamp Viral RNA Mini Kit, Qiagen, Venlo, Netherlands). Cells were then cultured for 3 weeks in 1 mg/ml G418 (Invitrogen, Carlsbad, CA, USA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Gaithersburg, MD, USA) supplemented with penicillin and streptomycin (100 U/ml) with 10% fetal bovine serum (Gibco) and grown at 37 °C in a humidified, 5% CO₂ atmosphere. siRNA reagents targeting V-ATPase (or non-specific siRNA) were purchased from Santa Cruz Biotechnology and delivered into A549 cells at a final concentration of 100 nM using the Amaxa nucleofection device (Buffer R, Program A-24, Lonza, Wokingham, UK) according to the manufacturer's instructions. Knockdown efficiency was then assessed by western blotting 24 h after transfection.

Lee M.R., Lee G.H., Lee H.Y., Kim D.S., Chung M.J., Lee Y.C., Kim H.R, & Chae H.J. (2014). BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation in pulmonary fibrosis. Cell Death & Disease, 5(3), e1113-.

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GILZ Knockdown Modulates Toxoplasma and Corticosterone Effects

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Knockdown of GILZ by siRNA was undertaken using siRNA reagents from Santa Cruz Biotechnology. MODE-K cells were grown in 6-well plates in DMEM media without antibiotics. Groups were as follows: Control (CON); Dexamethasone (DEX, 10⁻⁷ M); Toxoplasma infection (TOXO); Corticosterone (CORT, 10⁻⁵ M); DEX plus positive siRNA (DEX-siRNA⁺); Toxoplasma-CORT; Toxoplasma plus negative siRNA (TOXO-siRNA⁻; Toxoplasma-siRNA⁺; CORT-siRNA⁺; Toxoplasma-siRNA⁻, Toxoplasma-siRNA⁻, and CORT-siRNA⁻. Positive siRNA and negative wells were first washed with siRNA transfection medium, and overlaid with 10 µM positive and negative siRNA duplexes. At this time, Toxoplasma tachyzoites were added to the infected groups along with the addition of DEX and CORT. Positive and negative siRNA treated cells were allowed to incubate for 5 hr, after which 1 ml normal DMEM (with antibiotics) was added to all groups, incubated for 10 hr, and cells collected for mRNA analysis as indicated above. All samples treated with CORT were exposed for 15 hr, which is similar to the DUR treatment.

Johnson S.L., Gopal R., Enriquez A, & Monroy F.P. (2014). ROLE OF GLUCOCORTICOIDS AND Toxoplasma gondii INFECTION ON MURINE INTESTINAL EPITHELIAL CELLS. Parasitology international, 63(5), 687-694.

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Silencing 1-α-Hydroxylase and VDR in BV2 Cells

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siRNA reagents against 1-α-hydroxylase and VDR were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). BV2 cells (5×10⁴) were plated in 24-well plates and transfected with 1-α-hydroxylase (20 µmol/L) or VDR (50 µmol/L) siRNA using DharmaFECT reagent (Dharmacon Inc, Chicago, IL, USA) according to the manufacturer's protocol. After 24 h, the culture medium was replaced with fresh supplemented medium, and the cells were cultivated for an additional 24 h.

Hur J., Lee P., Kim M.J, & Cho Y.W. (2014). Regulatory Effect of 25-hydroxyvitamin D3 on Nitric Oxide Production in Activated Microglia. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 18(5), 397-402.

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