Control sirna

Manufactured by Eurogentec

Sourced in France, Belgium

Control siRNA is a laboratory reagent used for evaluating the effectiveness and specificity of small interfering RNA (siRNA) experiments. It serves as a non-targeting control to help determine the effects of siRNA transfection in cells, without directly affecting the expression of any specific gene.

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5 protocols using control sirna

Asymmetric Duplex siRNA Targeting TNF-α

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All chemicals were purchased from Acros, Aldrich, or Fluka. All solvents were freshly dried and distilled according to routine procedures before use. Asymmetric duplex siRNA directed against TNF-α fluorescent dicer substrate siRNA labeled with FAM (fluorescein amidite), and the negative control sequence were purchased from Eurogentec (Eurogentec, Angers, France) as dried, purified, and desalted duplexes. The oligonucleotide sequences in the anti-TNF-α A w f w g: sense 5'-GAC-AAC-CAA-CUA-GUG-GUG-cdtdt-3', anti-sense 5'-GCA-CCA-CUA-GUU-GGU-UGU-cdtdt-3'.
A negative control siRNA with the same number of bases as anti-TNF-α A w .
The exact sequence was not provided by the supplier (Eurogentec). Dulbecco's phosphate buffer saline, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT), hydrogen chloride (HCl), sodium hydroxide (NaOH), and lipopolysaccharide from Escherichia coli O111:B4 ( P ) (γ-irradiated, < 1 % protein) were obtained from Sigma-Aldrich (St Quentin Fallavier, France). Lactate dehydrogenase (LDH) kit was acquired from Promega (Charbonnières-Les-Bains, France). RNase-free water (Sigma-Aldrich, St Quentin Fallavier, France) was used for all solutions containing siRNA and dendrimers. All additional chemicals used were of analytical grade.

Yu Z., Tsapis N., Fay F., Chen L., Karpus A., Shi X., Cailleau C., García Pérez S., Huang N., Vergnaud J., Mignani S., Majoral J.P, & Fattal E. (2023). Amphiphilic Phosphorus Dendrons Associated with Anti-inflammatory siRNA Reduce Symptoms in Murine Collagen-Induced Arthritis. Biomacromolecules, 24(2).

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Antibody reagents for NF-kB signaling

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The following antibodies were used in this study: BCL10 (rabbit, sc-5611), BCL10 (goat, sc-9560), IKKα (H-744, sc-7218), IKKα/β (H-470, sc-7607), MALT1 (H-300, sc-28246), NEMO (FL-419, sc-8330), RelB (sc-226), Cylindromatosis-1/CYLD1 (E-10, sc-74435) were from Santa Cruz Biotechnology (Santa Monica, CA, USA). IκBα (44D4, #9242), CARD11/CARMA1 (1D12, #4435), GSK3β (27C10, #9315), GSK3α (#9338) were obtained from Cell Signaling (Danvers, MA, USA). ImmunoCult^TM Human CD3/CD28 T cell activator (#10971) was obtained from STEMCELL technologies Inc. (Cologne, Germany). Polyclonal Goat Anti-Mouse Ig (#553998) and Purified Mouse anti-β-catenin (#610154) were obtained from BD Biosciences (San Jose, CA, USA). β-Tubulin (TUB 2.1, #T4026), the GSK3β inhibitors SB216763 (Sigma-Aldrich, #S3442) and SB415286 (Sigma-Aldrich, #S3567), Cycloheximid (Sigma Aldrich, #C7698), and ionomycin (Sigma Aldrich, #I0634) were from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Control siRNA (#SR-CL000-005), GSK3βsiRNA1 (5′-GACUAGAGGGCAGAGUAAAU-3′) and GSK3βsiRNA2 (5′-CCGGGAACAAAUCCGAGAGAU-3′) were obtained from Eurogentec (Liege, Belgium). PMA was purchased (#524400) from Merck (Darmstadt, Germany).

Abd-Ellah A., Voogdt C., Krappmann D., Möller P, & Marienfeld R.B. (2018). GSK3β modulates NF-κB activation and RelB degradation through site-specific phosphorylation of BCL10. Scientific Reports, 8, 1352.

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Plasmid Transfection and AKT1 Modulation

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Plasmid pcDNA3 flag HA AKT1 was a gift from William Sellers (Addgene plasmid # 9021; http://n2t.net/addgene:9021; RRID: Addgene_9021) (28 (link)). The pcDNA3-HA-AKT1-K179M expression vector expressing an inactive mutant form of AKT1 (altered kinase domain) was a gift from Jie Chen (Addgene plasmid #73409; http://n2t.net/addgene:73409; RRID: Addgene_73409) (29 (link)). Plasmid mCherry-AKT1 E17K, causing constitutive activation of AKT1, was a generous gift from Dr Anne-Laure Todeschini. Cells were transfected with Jet Optimus Reagent (Polyplus Transfection Ref: 117-01).
With the AKT1 inhibitor, Lipofectamine 3000 (Life Technologies) was used according to the supplier's recommendations. ICAFectin™ 442 reagent (In Cell Art, Nantes, France) was used to transfect HEK293FT WT and ΔAKT1 cells with a control siRNA (Eurogentec), siRNA UPF1 (5′-AAGATGCAGTTCCGCTCCATTTT-3′), siRNA UPF2 (5′-GAAGTTGGTACGGGCACTC-3′), siRNA UPF3X (5′-GGAGAAGCGAGTAACCCTG-3′) (Sigma Aldrich), siRNA AKT1 (5′-GAAGGAAGUCAUCGUGGCCAA-3′), siRNA AKT2 (5′- CUCUUCGAGCUCAUCCUCA-3′), siRNA AKT3 (5′-GAAAGAUUGUGUACCGUGA-3′), siRNA SMG1 (5′- CCAGGACACGAGGAAACUG-3′) and pmCMV-Gl Norm or pmCMV-Gl Ter and pIE-MUP].

Palma M., Leroy C., Salomé-Desnoulez S., Werkmeister E., Kong R., Mongy M., Le Hir H, & Lejeune F. (2021). A role for AKT1 in nonsense-mediated mRNA decay. Nucleic Acids Research, 49(19), 11022-11037.

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RUVBL-1 Knockdown Procedure

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Ana2014 cells were transfected with either 200 nM control siRNA or 200 nM RUVBL-1 targeting siRNAs (Kaneka Eurogentec S.A.) as described previously (36 (link)). The three siRNA sets were used (Table S2). Then, 24 h posttransfection, cells were collected and used to perform qRT-PCR, Western blotting, and immunofluorescence studies as described above.

Araveti P.B., Kar P.P., Kuriakose A., Sanju A., Kumar K.A, & Srivastava A. (2023). Identification of a Novel Interaction between Theileria Prohibitin (TaPHB-1) and Bovine RuvB-Like AAA ATPase 1. Microbiology Spectrum, 11(1), e02502-22.

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Transfection of human and mouse macrophages

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Small interfering RNA (siRNA) transfection of human monocyte–derived macrophages (hMDMs) was as previously described by Marion et al. (24 (link)). Briefly, macrophages at d 4–5 were washed twice and kept in complete medium at 37°C. The siRNA solution was prepared in OptiMEM medium (GlutaMax supplemented; Thermo Fisher Scientific) containing Lipofectamine RNAiMax reagent (Thermo Fisher Scientific) and siRNA at a final concentration of 240 nM. Cells were incubated at 37°C for the indicated time. siRNA were 5′-GGCUGUAGGAAAUCUAGUU-3′ for NME1 and control siRNA 5′-CGUACGCGGAAUACUUCGA-3′ for Luciferase (Eurogentec, Liège, Belgium).
Five-day–matured bone marrow–derived macrophages (BMDMs) were transfected with On-Targetplus Smartpool siRNA specific for mouse Nme1 and On-TargetPlus Nontargeting Control Pool (Dharmacon, Lafayette, CO, USA) using the Dharmafect 1 Transfection Reagent (Dharmacon) according to Dharmafect’s Transfection Protocol. At 48 h after transfection, cells were harvested for detecting the protein level of Nme1 of transfected BMDMs by Western blot analysis.

Farkas Z., Petric M., Liu X., Herit F., Rajnavölgyi É., Szondy Z., Budai Z., Orbán T.I., Sándor S., Mehta A., Bajtay Z., Kovács T., Jung S.Y., Afaq Shakir M., Qin J., Zhou Z., Niedergang F., Boissan M, & Takács-Vellai K. (2019). The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin. The FASEB Journal, 33(10), 11606-11614.

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