Sirt1 sirna

Manufactured by Thermo Fisher Scientific

Sourced in United States

SIRT1 siRNA is a laboratory reagent designed for gene silencing experiments. It is a small interfering RNA (siRNA) that targets the SIRT1 gene, which encodes the sirtuin 1 protein. This product can be used to study the biological functions of SIRT1 in various cell-based models.

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17 protocols using sirt1 sirna

Silencing Sirt1 in HEK293 Cells

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HEK293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in 10% fetal bovine serum (FBS) and 1% PenStrep‐supplemented Dulbecco's Modified Eagle Medium (DMEM) medium. Sirt1 SiRNA (siRNA ID.136459, Catalog #AM16708, ThermoFisher) was used to transfect 293 cells. Briefly, Sirt1 SiRNA was diluted to 100 nM in serum‐free DMEM and Lipofectamine 2000 (Invitrogen) and was incubated at room temperature for 15 min. Cells were transfected for 6–8 h and used for subsequent experiments within 48 h.

Yoon J., Daneshgar N., Chu Y., Chen B., Hefti M., Vikram A., Irani K., Song L., Brenner C., Abel E.D., London B, & Dai D. (2022). Metabolic rescue ameliorates mitochondrial encephalo‐cardiomyopathy in murine and human iPSC models of Leigh syndrome. Clinical and Translational Medicine, 12(7), e954.

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Knockdown of ATG5 and Sirt1 in SH-SY5Y Cells

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SH-SY5Y cells were transfected with ATG5 small interfering RNA (siRNA; oligoID HSS114104; Invitrogen, Carlsbad, CA, USA) or Sirt1 siRNA (oligoID VHS50608; Invitrogen) using Lipofectamine 2000 according to manufacturer's instructions. After a 48-hr culture, knockdown efficiency was measured at the protein level by immunoblot analysis. Nonspecific siRNA (oligoID 12935-300; Invitrogen) was used as the negative control.

Lee J.H., Moon J.H., Kim S.W., Jeong J.K., Nazim U.M., Lee Y.J., Seol J.W, & Park S.Y. (2015). EGCG-mediated autophagy flux has a neuroprotection effect via a class III histone deacetylase in primary neuron cells. Oncotarget, 6(12), 9701-9717.

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Modulating miR-29b and SIRT1 in CRC

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Mature human miR-29b (5′-UAGCACCAUU UGAAAUCAGUGUU-3′), negative control oligon ucleotides (NCO, 5′-AUAUAGUCAUGUUCCAC UAGUG-3′), 2’-Omethyl modified miR-29b inhibitors (anti-miR-29b, 5′-AACACUGA UUUCAAAUGGUGCUA-3′) and SIRT1 siRNA (forward: 5′-GAUGACGUCUUAUCCUCUAUU-3′, reverse: 5′-UAGAGGAUAAGACGUCAUCUU-3′) were purchased from RiboBio Co. Ltd (Guangzhou, China). For overexpression of SIRT1, open reading frame region of human SIRT1 was reconstructed with pcDNA3.1 eukaryotic expression plasmid (Invitrogen, USA). For transfection, 50 pmol/ml miR-29b, 50 pmol/ml anti-miR-29b, 50 pmol/ml NCO, 50 pmol/ml SIRT1 siRNA or 2 μg/ml SIRT1 plasmid was transfected into CRC cells by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Liu H, & Cheng X.H. (2018). MiR-29b reverses oxaliplatin-resistance in colorectal cancer by targeting SIRT1. Oncotarget, 9(15), 12304-12315.

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Sirt1 and Nrf2 Knockdown in Glomerular Mesangial Cells

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The specific siRNA sequences, including Sirt1-siRNA, Nrf2-siRNA and the negative control, were synthesized by Sangon Biotech (Shanghai, China). The sequences of Sirt1-siRNA were as follows: sense: 5′-CCAGUAGCACUAAUUCCAATT-3′, antisense: 5′-UUGGAAUUAGUGCUACUGGTT-3′. The sequences of Nrf2-siRNA were as below: sense: 5′-GAGGAUGGGAAACCUUACUTT-3′, antisense: 5′-AGUAAGGUUUCCCAUCCUCTT-3′. Appropriate siRNA was transiently transfected into GMCs with lipofectamine^® RNAiMAX (Invitrogen, United States). After 48 h incubation, the transfection medium was replaced with fresh serum-free DMEM for another 12 h. After various treatments, the cells were harvested for WB.

Zhang L., Chen Z., Gong W., Zou Y., Xu F., Chen L, & Huang H. (2018). Paeonol Ameliorates Diabetic Renal Fibrosis Through Promoting the Activation of the Nrf2/ARE Pathway via Up-Regulating Sirt1. Frontiers in Pharmacology, 9, 512.

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Isogenic NSCLC Cell Line Generation

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Human NSCLC cell lines A549 and H596 were purchased from the American Type Culture Collection (ATCC, USA) and isogenically matched NQO1-expressing H596 cells were generated by infecting with lentiviral vectors expressing NQO1 or empty vector. LAT1 plasmid was kindly provided by Prof. Chen L from Tsinghua University (Beijing, China). Cells were transfected with FOXO1, NQO1 siRNA, SIRT1 siRNA or LAT1 siRNA (Invitrogen) using lipofectamine™ RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's reverse transcription protocol. FOXO1, NQO1 and LAT1 siRNA sequences are shown in Supplementary Table S1.

Liu H., Xing R., Cheng X., Li Q., Liu F., Ye H., Zhao M., Wang H., Wang G, & Hao H. (2016). De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells. Oncotarget, 7(38), 62503-62519.

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Modulation of miR-217 and SIRT1 in THP-1 Macrophages

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miR-217 inhibitor (5′-UACUGCAUCAGGAACUGAUUGGA-3′; Shanghai GenePharma Co., Ltd.), inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Shanghai GenePharma Co., Ltd.), control-siRNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.), SIRT1-siRNA (cat. no. sc-40986; Santa Cruz Biotechnology, Inc.) or miR-217 inhibitor + SIRT1-siRNA was transfected into THP-1 macrophages (ox-LDL untreated) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol at 37°C for 48 h. Transfection efficiency was measured at 48 h post-transfection using reverse transcription-quantitative PCR (RT-qPCR).

Zhang L., Chen J., He Q., Chao Z., Li X, & Chen M. (2019). MicroRNA-217 is involved in the progression of atherosclerosis through regulating inflammatory responses by targeting sirtuin 1. Molecular Medicine Reports, 20(4), 3182-3190.

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Plasmid and siRNA Transfection Protocols

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For plasmid transfection studies, the cells were transfected with control, Flag-FXR WT/mutants, Flag-CHIP WT/mutants, CHIP WT/mutants, GFP-FXR WT/mutants, Flag-SIRT1, GFP-NLS Cargo using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's protocols.
For siRNA transfection studies, the cells were transfected with control siRNA, NEDD4 siRNA, ITCH siRNA, CHIP siRNA, BARD1 siRNA, SMURF1 siRNA (Nanjing EKBIO Technology), CRM1 siRNA (Santa Cruz), SIRT1 siRNA (Invitrogen) using lipofectamine RNAiMAX reagent (Invitrogen) for 48 h before ActD/TNFα treatment, according to the manufacturer's protocols.

Cui S., Hu H., Chen A., Cui M., Pan X., Zhang P., Wang G., Wang H, & Hao H. (2022). SIRT1 activation synergizes with FXR agonism in hepatoprotection via governing nucleocytoplasmic shuttling and degradation of FXR. Acta Pharmaceutica Sinica. B, 13(2), 559-576.

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Modulation of SIRT1 Expression in Prostate Cancer

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Mature human miR-204 (5’-UUCCCUUUGUCAUCCUAUGCCU-3’) and negative control oligonucleotides (NCO, 5’-UCUCUUUAUCCUGUCUACCUGC-3’) were purchased from GenePharma Co. Ltd (Shanghai, China). SIRT1 small interfere RNA (siRNA) was purchased from Santa Cruze Biotechnology (USA). For enforced expression of SIRT1 in PCa cells, open reading frame region of human SIRT1 gene was amplified and inserted into the pcDNA3.1 eukaryotic expression vector (Invitrogen, USA). Before transfection, cells were seeded into 6-well plates overnight. 50 pmol/ml miR-204, NCO and SIRT1 siRNA, or 2 μg/ml SIRT1 plasmid was then transfected into the PCa cells by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Shu Y., Ren L., Xie B., Liang Z, & Chen J. (2017). MiR-204 enhances mitochondrial apoptosis in doxorubicin-treated prostate cancer cells by targeting SIRT1/p53 pathway. Oncotarget, 8(57), 97313-97322.

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Modulating SIRT1 and miR-140 Expression

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To overexpress SIRT1, SIRT1 expression vector was conducted by cloning the open reading frame of SIRT1 gene into the pcDNA3.1 plasmid (Invitrogen, CA, USA). To directly knockdown SIRT1, SIRT1 small interfere RNA (siRNA) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). To overexpress miR-140, miR-140 mimic was purchased from GenePharma Co. Ltd. (Shanghai, China). To knockdown miR-140, miR-140 antisense oligonucleotide (anti-miR-140) was purchased from GenePharma Co. Ltd. To perform the gain (loss)-of-function experiments of miR-140 and SIRT1, cells were transient transfected with miR-140 (50 pmol/mL), anti-miR-140 (50 pmol/mL), SIRT1 plasmid (2 μg/mL) or SIRT1 siRNA (50 pmol/mL) by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Negative control oligonucleotides (NCO, Genechem Co., Ltd), empty pcDNA3.1 plasmid and control siRNA were used as internal control.

Lin Z., Pan J., Chen L., Wang X, & Chen Y. (2020). MiR-140 Resensitizes Cisplatin-Resistant NSCLC Cells to Cisplatin Treatment Through the SIRT1/ROS/JNK Pathway. OncoTargets and therapy, 13, 8149-8160.

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Investigating miR-217 and SIRT1 in OGD/R

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Cortical neurons were seeded in a 6-well plate (1×10⁶ cells/well) and incubated at 37°C for 24 h. Then, 100 nM miR-217 inhibitor (5′-UACUGCAUCAGGAACUGAUUGGA-3′; Bioneer Corp.), 100 nM inhibitor control (5′-GCCUCCGGCUUCGCACCUCU-3′; Bioneer Corp.), 2 µl control-small interfering RNA (siRNA; cat. no. sc-36869; Santa Cruz Biotechnology, Inc.), 2 µl sirtuin 1 (SIRT1)-siRNA (cat. no. sc-40986; Santa Cruz Biotechnology, Inc.) or 100 nM miR-217 inhibitor + 2 µl SIRT1-siRNA was transfected into the cells using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. At 24 h after transfection, transfection efficiency was measured using reverse transcription-quantitative PCR (RT-qPCR). At 24 h following transfection with miR-217 inhibitor, inhibitor control, or miR-217 inhibitor+SIRT1-siRNA, cells were subjected to OGD/R induction.

Rao G., Zhang W, & Song S. (2019). MicroRNA-217 inhibition relieves cerebral ischemia/reperfusion injury by targeting SIRT1. Molecular Medicine Reports, 20(2), 1221-1229.

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