Control sirna

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany, China

Control siRNA is a laboratory tool used to verify the specificity of RNA interference (RNAi) experiments. It is a non-targeting siRNA sequence that does not match any known gene in the target organism's genome. This control siRNA is designed to have no known biological effects, serving as a reference to distinguish target-specific effects from non-specific or off-target effects during RNAi studies.

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TEAD1 siRNA and controls Control siRNAs (sc-37007) Random control siRNA Control nonspecific siRNA oligonucleotides Control siRNA (siControl) Non-specific control siRNA Control siRNA (sc-37007) Nonspecific control siRNA oligonucleotide Control siRNA (si-C) Control siRNA duplex

510 protocols using control sirna

Silencing ASK1 in HT-29 Cells

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Control siRNA and siRNA targeting ASK1 were obtained from Santa Cruz Biotechnology, Inc. The sense sequences of the siRNA reagents were 5′-GACGCGATCAGAGAGTAAT-3′ (siRNA control) and 5′-GGTGGCACAAGCAAGTTCT-3′ (siRNA ASK1). For transient siRNA transfection, HT-29 cells were seeded at a density of 5×10⁵ cells/ml into six-well plates. Cells were transfected on the following day with the Lipofectamine^® LTX with Plus™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 100 ng/well siRNA (ASK1 or control). Cells were transfected with each siRNA and incubated for 48 h. The interference of ASK1 protein expression was confirmed by western blot analysis using the anti-ASK1 antibody.

QU L., LIU F.X., CAO X.C., XIAO Q., YANG X, & REN K.Q. (2014). Activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase pathway is involved in the casticin-induced apoptosis of colon cancer cells. Experimental and Therapeutic Medicine, 8(5), 1494-1500.

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Transient Knockdown of ROCK1 and Ezrin

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For transient knockdown experiments, cells were transfected with ROCK1-specific siRNA (ROCK1 siRNA) or control siRNA, and ezrin specific siRNA (ezrin siRNA) or control siRNA (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively. Cells were first plated onto 6-well plates. ROCK1 siRNA, ezrin siRNA or control siRNA duplexes were diluted to a final concentration of 10 μmol/L in OptiMem (Invitrogen, San Diego, CA, USA) and incubated with Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 15 min. The mixture was then incubated with the cells under serum- and antibiotic-free conditions for 18 h at 37 °C. Cells were then washed twice with sterile PBS and incubated in William's E medium supplemented with 5% calf serum for 24 h prior to exposure to LPS.

Ding N., Li P., Li H., Lei Y, & Zhang Z. (2022). The ROCK-ezrin signaling pathway mediates LPS-induced cytokine production in pulmonary alveolar epithelial cells. Cell Communication and Signaling : CCS, 20, 65.

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Silencing Glucocorticoid Receptor in Osteoclasts

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For in vitro siRNA transfection, GR siRNA (Santa Cruz Biotechnology; sc-35506) and Control siRNA (Santa Cruz Biotechnology; sc-37007) were transfected into POCs according to the manufacturer’s guidelines. Briefly, RAW264.7 cells were induced to differentiate into POCs by culture for 3 days in the presence of RANKL and M-CSF. POCs were transfected with either GR siRNA or Control siRNA duplex diluted in siRNA transfection medium (Santa Cruz Biotechnology; sc-36868) and mixed with siRNA transfection reagent (Santa Cruz Biotechnology; sc-29528) into siRNA transfection medium after incubation for 30 min at room temperature. Cells were incubated for 6 hours at 37°C in a 5% CO₂ incubator. Transfection mixture was removed and replaced with Dulbecco’s Modified Eagle Medium (DMEM) culture medium containing 10% FBS, RANKL 60 ng/mL, and M-CSF 30 ng/mL, and cells were incubated for an additional 24 hours. Conditioned medium, chromatin, cell protein, and RNA were collected for ELISA, chromatin immunoprecipitation (ChIP), Western blot analysis, and RT-PCR.

Peng Y., Lv S., Li Y., Zhu J., Chen S., Zhen G., Cao X., Wu S, & Crane J.L. (2020). Glucocorticoids Disrupt Skeletal Angiogenesis Through Transrepression of NF-κB–Mediated Preosteoclast Pdgfb Transcription in Young Mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 35(6), 1188-1202.

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Gene Manipulation for Cell Line

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GLUT1 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (CA, USA; catalog number: sc-35493), integrin β1 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (CA, USA; catalog number: sc-35674), and GLUT1 plasmid were purchased from OriGene (Rockville, USA; catalog number: SC116011). They were transiently transfected to cell line with Lipofectamine 2000 (Invitrogen, Carlsbad, USA; catalog number: 11668030) according to the instruction of manufacturer.

Zhao H., Sun J., Shao J., Zou Z., Qiu X., Wang E, & Wu G. (2019). Glucose Transporter 1 Promotes the Malignant Phenotype of Non-Small Cell Lung Cancer through Integrin β1/Src/FAK Signaling. Journal of Cancer, 10(20), 4989-4997.

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Modulation of miR-375 and its Targets in Macrophages

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Both miR mimic and siRNA transfection were performed using HiPerfect (Qiagen) according to the manufacturer’s instructions. For overexpression of miR-375 primary human MΦ in six-well plates were transfected with MISSION^® hsa-miR-375 mimic or MISSION^® miR negative control 2 from C. elegans (cel-miR-39a; both from Sigma-Aldrich). De novo synthesis of mature miRs in MΦ upon coculture was prevented using DICER siRNA or control siRNA (Santa Cruz biotechnology). To block CD36 gene expression, MΦ were transfected with ON-TARGETplus CD36 siRNA or control siRNA (both from Dharmacon, Lafayette, Colorado, USA). Double KD of PXN and TNS3 was performed in primary human MΦ using PXN/TNS3 siRNA or control siRNA (Santa Cruz biotechnology). To interfere with the binding of miR-375 to its targets, miRCURY LNA^TM miRNA Power Target Site Blockers (Qiagen) with the following custom sequences were used according to the manufacturer’s instructions: PXN 5′-CTGTCCATCCCGCACCAGCG-3′, TNS3 5′-CTCGCCCAGCTCGCCCA-3′, and scramble control 5′-ACGTCTATACGCCCA-3′.

Frank A.C., Ebersberger S., Fink A.F., Lampe S., Weigert A., Schmid T., Ebersberger I., Syed S.N, & Brüne B. (2019). Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype. Nature Communications, 10, 1135.

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Silencing KRAS and HSP90 in Colorectal Cancer

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HCT116 p53 (+/+) and p53 (−/−) cells were seeded in 35 mm plates at 300,000 cells per well. Twenty-four hours later, cells were transfected with 80 nM KRAS Silencer Select Pre-Designed & Validated siRNA (siRNA KRAS) or 80 nM Silencer Negative Control siRNA (siRNA control) (both from Applied Biosystems, Foster City, CA, USA). In parallel SW620 cells were seeded in 35 mm plates at 300,000 cells per well. Twenty four hours later, cells were transfected with 50 or 100 nM siRNA KRAS, siRNA control, siRNA HSP90 (HSP90α/β siRNA (h) (sc-35608 Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and co-transfected with 50 nM siRNA KRAS plus 50 nM siRNA HSP90. Transfections were performed using lipofectamine 3000 (Invitrogen), according to manufacturer’s instructions. Twenty-four h later, cells were replated in 24-well plates at 50,000 cells/well and 72 h later Guava ViaCount, trypan blue dye exllusion, MTS metabolism and LDH release assays.

Brito H., Martins A.C., Lavrado J., Mendes E., Francisco A.P., Santos S.A., Ohnmacht S.A., Kim N.S., Rodrigues C.M., Moreira R., Neidle S., Borralho P.M, & Paulo A. (2015). Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives. PLoS ONE, 10(5), e0126891.

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Modulating miR-224 and miR-663 in Cells

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MiR-224 or miR-663 inhibitors (anti-miR-224 or anti-miR-663), miR-663 mimics and their negative control oligonucleotides (anti-miR-NC or miR-NC mimics) were obtained from Ambion Inc. (Austin, TX, USA). siRNA/p21 (sc-29428) (Santa Cruz Biotechnology, Santa Clara, CA, USA) or siRNA/control was purchased from Santa Cruz Inc. (Santa Clara, CA, USA). The open reading frame of p21 that was generated by PCR was then inserted into the pcDNA 3.1 expression vector, which was named pcDNA/p21. The recombinant vector was confirmed by the digestion analysis of restriction endonuclease and DNA sequencing. For ectopic expression of miR-224, the pGCMV/miR-224 or pGCMV/miR-NC vectors were purchased from GenePharm (Shanghai, China). The transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the instructions provided by the manufacturer. The cells were transfected with those recombinant DNA vectors containing a G418 selection marker and were selected by G418 (Sigma, St Louis, MO, USA) at 500 mg ml⁻¹ for 4 weeks Then, single clones were obtained and maintained in G418 with concentration of 200 mg ml⁻¹.

Wang H., Zhu L.J., Yang Y.C., Wang Z.X, & Wang R. (2014). MiR-224 promotes the chemoresistance of human lung adenocarcinoma cells to cisplatin via regulating G1/S transition and apoptosis by targeting p21WAF1/CIP1. British Journal of Cancer, 111(2), 339-354.

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MEL Cell Culture Protocol

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MEL cells were donated by Dr. Crispino’s lab. All cell culture media, supplements, and buffers were from Thermo Fisher (Waltham, MA). Vacuolin-1 was obtained from Millipore (Burlington, MA). ORC4 antibodies, siRNA-Control, and siRNA-ORC4 plasmids were purchased from Santa Cruz Biotechnology (Dallas, Texas). Edu staining kit and Lipofectamin-^2000TM were purchased from Thermo Fisher (Waltham, MA).

Nguyen H., Ung A, & Ward W.S. (2020). The Role of ORC4 in Enucleation of Murine Erythroleukemia (MEL) Cells is Similar to that in Oocyte Polar Body Extrusion. Systems biology in reproductive medicine, 66(6), 378-386.

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Transient Overexpression of miRNAs, siRNAs, and Plasmids

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For transient overexpression of miRNAs and siRNAs, cells at 50% confluence were transfected using Oligofectamine (Invitrogen, Milan, Italy) and 100 nM of pre-miR-340, scrambled miRNA, anti-miR-340, scrambled anti-miRNA (Ambion^®, Life Technologies), siNRAS or a siRNA control (Santa Cruz Biotechnologies, MA, USA). For transient overexpression of 4 μg of pcDNA3-NRAS, pcDNA3-AKT⁺, pcDNA3-ERK⁺ or pcDNA3, cells were transfected using X-tremeGENE 9 DNA Transfection Reagent (Roche, Milan, Italy). Temozolomide (TMZ) was purchased from Sigma Aldrich (Milan, Italy).

Fiore D., Donnarumma E., Roscigno G., Iaboni M., Russo V., Affinito A., Adamo A., De Martino F., Quintavalle C., Romano G., Greco A., Soini Y., Brunetti A., Croce C.M, & Condorelli G. (2016). miR-340 predicts glioblastoma survival and modulates key cancer hallmarks through down-regulation of NRAS. Oncotarget, 7(15), 19531-19547.

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siRNA Knockdown of EGFR, Atg7, and CK2α

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Small interfering RNA (siRNA) oligonucleotides specific for EGFR, Atg7, CK2α and the siRNA control were purchased from Santa Cruz Biotechnology (Santa Cruz). Cells were seeded into a 60-mm dish which was then left for 24 h. A 2 µL aliquot of siRNA solution (10 µM) and 5 µL of Lipofectamine 2000 (Invitrogen) were each mixed with 100 µL of serum-free RPMI 1640 medium. They were incubated for 20 min at room temperature after combining the two mixtures, and this was then added to the cells that had been seeded on the dish. After 24 h, harvested cells were subjected to Western blot analysis. Cells were also processed for cell viability and apoptosis analysis.

So K.S., Kim C.H., Rho J.K., Kim S.Y., Choi Y.J., Song J.S., Kim W.S., Choi C.M., Chun Y.J, & Lee J.C. (2014). Autophagosome-Mediated EGFR Down-Regulation Induced by the CK2 Inhibitor Enhances the Efficacy of EGFR-TKI on EGFR-Mutant Lung Cancer Cells with Resistance by T790M. PLoS ONE, 9(12), e114000.

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