Nc sirna

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

NC siRNA is a non-coding small interfering RNA (siRNA) used as a negative control in gene knockdown experiments. It is designed to have no known target in the tested species and serves as a reference to distinguish specific gene silencing effects from non-specific effects.

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ON-TARGET plus siRNA or NCs (2 citations) Non-specific siRNA (si-NC) (7 citations) SiRNAs (258 citations)

113 protocols using nc sirna

Bladder Cancer Cell Line Transfection with XIST and miR-133a

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The SV-HUC-1 normal urinary tract epithelial cell line and four common bladder cancer cell lines, T24, 253J, RT112 and HT-1376, were obtained from the Cell Bank of the Chinese Academy of Sciences. These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% foetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. For cell transfection, 253J and RT112 cells were transfected with 100 nM XIST small interfering (si)RNA1 (5′-GCAAAUGAAAGCUACCAAU-3′; Thermo Fisher Scientific, Inc.), XIST siRNA2 (5′-GCACAAUAUCUUUGAACUA-3′; Thermo Fisher Scientific, Inc.) or 100 nM negative control (NC) siRNA (cat. no. 4459405; Thermo Fisher Scientific, Inc.) or co-transfected with 100 nM NC siRNA and 100 nM NC inhibitor (cat. no. 4464076; Thermo Fisher Scientific, Inc.), or 100 nM NC siRNA and 100 nM miR-133a inhibitor (cat. no. 4464084; Thermo Fisher Scientific, Inc.), or 100 nM XIST siRNA and 100 nM miR-133a inhibitor, or 100 nM XIST siRNA and 100 nM NC inhibitor using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. At 48 h after transfection, the cells were harvested and used for the subsequent assays.

Zhou K., Yang J., Li X, & Chen W. (2019). Long non-coding RNA XIST promotes cell proliferation and migration through targeting miR-133a in bladder cancer. Experimental and Therapeutic Medicine, 18(5), 3475-3483.

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Silencing lncRNA ASAP1-IT1 in HCC Cells

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Silencing of lncRNA ASAP1-IT1 was performed using ASAP1-IT1-specific small interfering RNA (siRNA) in HCC cell lines. ASAP1-IT1-specific siRNA and non-specific control siRNA (NC-siRNA) were synthesized by Invitrogen (Carlsbad, CA, USA), and their sequences were as follows: ASAP1-IT1-specific siRNA1: 5’-GCUGCGACAAUAGACAUCGGAGUUU-3’; ASAP1-IT1-specific siRNA 2: 5’-CAGCACCCGAUGUCAUUCCUGGGAA-3’; ASAP1-IT1-specific siRNA3: 5’-UGAAGGCAGAGUGGUAGGCUCGGAA-3’; NC siRNA: 5’-UUCUCCGAACGUGUCACGUTT-3’. In brief, HCC cells were initially seeded in a 24-well plate. After reaching 70-80% confluency, the cells were transfected with ASAP1-IT1-specific siRNA or NC-siRNA using the transfection reagent Lipofectamine^® 2000 (Invitrogen, USA) according to the manufacturer’s instructions. At 48 h after transfection, the cells were harvested for subsequent analysis.

Liu Y., Hu C., Qu X., Chen H., Liu L., Zhou L., Liu S., Li G, & Zhou Y. (2022). Novel Role of Long Non-Coding RNA ASAP1-IT1 in Progression of Hepatocellular Carcinoma. Frontiers in Oncology, 12, 746896.

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Ski-siRNA Knockdown in MG-63 and U2OS Cells

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Ski specific siRNA (Ski-siRNA) and scrambled sequence siRNA (negative control siRNA, NC-siRNA) were purchased from Thermo Fisher Scientific. The target sequence of Ski-siRNA was 5′-CGGACCTTGGCTGGTTCCTCCAATA-3′, and the sequence of 5′-TTCTCCGAACGTGTCACGT-3′ was considered to be NC-siRNA. The MG-63 and U2OS cells were transfected with Ski-siRNA or NC-siRNA using Lipofectamine^® RNAiMAX Reagent (Thermo Fisher Scientific) in accordance with the manufacture's protocol. Following 48 h of transfection, the cells were collected for the used in the following experiments. Western blot analyses were used to assess the silencing effect of Ski-siRNA. The cells were pre-treated with basic culture medium DMEM/F12 with or without 50 µM LY294002 (a specific PI3K inhibitor, S1737, Beyotime) for 6 h prior to transfection.

Zhao X., Fang Y., Wang X., Yang Z., Li D., Tian M, & Kang P. (2019). Knockdown of Ski decreases osteosarcoma cell proliferation and migration by suppressing the PI3K/Akt signaling pathway. International Journal of Oncology, 56(1), 206-218.

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Silencing DNMT1 Expression in 95D Cells

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95D cells were seeded in 24-well plates (1.5×10⁵ cells/well) and incubated for 24 h in a humidified atmosphere at 37°C with 5% CO₂. Negative control (NC) siRNA was purchased from Ambion (Thermo Fisher Scientific, Inc.). DNMT1 siRNA was constructed as described previously (20 ). NC siRNA and DNMT1 siRNA were transfected into the cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following incubation for at 37°C for 48 h, 95D cells transfected with NC siRNA or DNMT1 siRNA were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to validate the siRNA knockdown.

Bu X., Zhang X., Xu J., Yang H., Zhou X., Wang H, & Gong L. (2018). Inhibition of DNA methyltransferase 1 by RNA interference reverses epithelial-mesenchymal transition in highly metastatic 95D lung cancer cells by inhibiting the Wnt signaling pathway. Oncology Letters, 15(6), 9242-9250.

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Silencing Mouse FXR using siRNA

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Small interfering RNA (siRNA) targeting mouse FXR (siRNA-FXR) and its negative control (siRNA-NC) were synthesized by GenePharma (Shanghai, China). The sequences of siRNA-FXR were 5′-CAGGUUUGUUAACUGAAAUTT-3′ (sense) and 5′-AUUUCAGUUAACAAACCUGTT-3′ (antisense). Being randomized into siRNA-FXR and siRNA-NC groups, the AML12 cells were transfected with siRNA-FXR or siRNA-NC using LIPOFECTAMINE 3000 (L3000015, Thermo Fisher Scientific) according to the instructions of the manufacturer. The effect of RNA interference on FXR was tested by reverse transcription-quantitative polymerase chain reaction (RT-QPCR) and Western blot.

Xie Y., Shen F., He Y., Guo C., Yang R., Cao H., Pan Q, & Fan J. (2023). Gamma-Muricholic Acid Inhibits Nonalcoholic Steatohepatitis: Abolishment of Steatosis-Dependent Peroxidative Impairment by FXR/SHP/LXRα/FASN Signaling. Nutrients, 15(5), 1255.

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Silencing PRL15 and Regulating miR-567 in AGS Cells

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PRL15-specific siRNA (si-PRL15) and negative control siRNA (siRNA NC) were designed and purchased from Invitrogen (USA). SiRNA-PRL15 or siRNA NC was transfected into AGS cells with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. miR-567 mimic, miR-567 inhibitor, and matched negative controls (NC mimic and NC inhibitor) were synthesized by GenePharma (Shanghai, China). AGS cells were transfected using Lipofectamine 2000 (Invitrogen, Waltham, Beijing, China) and harvested after 48 h.

Ma Y., Xue H., Wang W., Yuan Y, & Liang F. (2020). The miR-567/RPL15/TGF-β/Smad axis inhibits the stem-like properties and chemo-resistance of gastric cancer cells. Translational Cancer Research, 9(5), 3539-3549.

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AS-IV Modulates Ferroptosis in PC12 Cells

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PC12 cells were incubated with 0, 0.01, 0.1, 1, 10, or 100 µM AS-IV (HPLC ≥98.0%, Cat No. JZ16042403) for 0, 24, 48, 72, and 96 h. Negative control (NC) small interfering (si)RNA and si-transcription factor EB (TFEB) were obtained from GenePharma (Shanghai, China). The H₂O₂-treated PC12 cell model was established using 300 µmol/L H₂O₂. The H₂O₂-damaged PC12 cells were treated with 0.1 µM AS-IV and 5 µM FIN56 (AbMole, USA), which is a specific inducer of ferroptosis. Cells were transfected with NC siRNA and si-TFEB using Lipofectamine 3000 (Life Technologies).

Zhou Y., Li L., Mao C, & Zhou D. (2022). Astragaloside IV ameliorates spinal cord injury through controlling ferroptosis in H2O2-damaged PC12 cells in vitro. Annals of Translational Medicine, 10(21), 1176.

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Silencing S100A4 in Vascular Smooth Muscle Cells

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The murine S100A4 siRNA (S100A4 siRNA) sequence was 5′-UGA ACA AGA CAG AGC UCA Att-3′ (sense) and 5′-UUG AGC UCU GUC UUG UUC Att-3′ (antisense), and the non-targeting siRNA (NC siRNA) sequence was sense: 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense: 5′-ACGUGACACGUUCGGAGAATT-3′ were purchased from Life Technologies (Shanghai, China). A7r5 cells were transfected with a final concentration of 20 nM of siRNA for 6 h performing in Lipofectamine™ 2000 according to the manufacturer’s recommendations. After incubation for 6 h, the medium was replaced with the standard culture medium. After an additional 42 h incubation, cells were used for further experiments. The transfected colonies referred to as A7r5/S100A4 siRNA or A7r5/NC siRNA cells, respectively.

Wang S., Li P., Jiang G., Guan J., Chen D, & Zhang X. (2020). Long non-coding RNA LOC285194 inhibits proliferation and migration but promoted apoptosis in vascular smooth muscle cells via targeting miR-211/PUMA and TGF-β1/S100A4 signal. Bioengineered, 11(1), 718-728.

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Knockdown of mTOR pathway components

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Down-regulation of target gene expression in cells was done by transfection of siRNA oligonucleotides with Lipofectamine 2000 transfection reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. A549 or PC-9 cells were plated on a 35-mm culture dish in RPMI-1640 complete growth medium to reach 50% ~ 60% confluence the next day when they were transfected with siRNAs as previously described[45 (link)]. The negative control (NC) siRNA and siRNAs against Raptor, Rictor, and MTOR were synthesized by Life Technologies. The sequences of siRNAs for Raptor, Rictor, and MTOR were indicated below: 5'-GGACAACGGCCACAAGUAC-3', 5'-GCGAGCTGATGTAGAATTG-3', and 5'-AACGCGGCACUUCACAAUUGA-3', respectively.

Jiang S., Zou Z., Nie P., Wen R., Xiao Y, & Tang J. (2015). Synergistic Effects between mTOR Complex 1/2 and Glycolysis Inhibitors in Non-Small-Cell Lung Carcinoma Cells. PLoS ONE, 10(7), e0132880.

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Silencing PLCγ-1 in A549 cells

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Small interfering RNAs (siRNA) targeting PLCγ-1 and a siRNA negative control were obtained from Qiagen (Valencia, CA, USA). A549 cells were mixed with 10 nM siRNA for PLCγ-1 or negative control siRNA (NC-siRNA) and then electroporated using the Neon Transfection System (Life Technologies Japan). After electroporation, cells were transferred to 24-well plates or a poly-L-lysine glass-bottom dish containing pre-warmed antibiotic-free media and incubated at 37°C. Experiments were performed 48 h after transfection. Silencing was verified by western blotting with anti-PLCγ-1 antibody (Cell Signaling Technology).

Takagishi T., Oda M., Kabura M., Kurosawa M., Tominaga K., Urano S., Ueda Y., Kobayashi K., Kobayashi T., Sakurai J., Terao Y, & Nagahama M. (2015). Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane. PLoS ONE, 10(4), e0120497.

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