Sirna design center

Manufactured by Thermo Fisher Scientific

Sourced in United States

The SiRNA Design Center is a tool provided by Thermo Fisher Scientific that enables the design of small interfering RNA (siRNA) sequences. The core function of this tool is to assist researchers in the selection of optimal siRNA sequences for gene silencing experiments. The SiRNA Design Center utilizes algorithms to analyze target gene sequences and generate potential siRNA candidates that meet specific design criteria.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Luciferase sirna, by Horizon Discovery (1 mentions)

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SiRNAs

4 protocols using sirna design center

Downregulation of PD-L1 Using siRNA-NPs

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All siRNAs were purchased from Dharmacon (Lafayette, CO, USA). Untreated cells and cells treated with non-targeting scrambled siRNA (Dharmacon, Catalog Item D-001810-10-20) were used as controls. Isoform‐specific siRNA was custom designed using Thermo Scientific siRNA Design Center (Thermo Scientific, Rockford, IL, USA). siRNA specific sequence was 5’-GAGGAAGACCUGAAGGUUCAGCAUA-3’ for PD-L1. For scrambled siRNA, we used the commercial ON-TARGETplus Non-targeting Control Pool (catalog number D-001810-10-20) comprised of the following siRNA sequences: 5’-UGGUUUACAUGUCGACUAA-3’, 5’-UGGUUUACAUGUUGUGUGA-3’, 5’-UGGUUUACAUGUUUUCUGA-3’ and 5’-UGGUUUACAUGUUUUCCUA-3’.
Cells were incubated for 48 h in RPMI 1640 medium containing siRNA-PD-L1 dextran NPs (concentration of siRNA: 100 pmol/mL, N/P = 15). Cells were treated with NPs for 48 h, because this incubation period resulted in the most effective downregulation of the target genes. All transfections were carried out based on established protocols (20 (link)).

Pacheco-Torres J., Penet M.F., Krishnamachary B., Mironchik Y., Chen Z, & Bhujwalla Z.M. (2021). PD-L1 siRNA Theranostics With a Dextran Nanoparticle Highlights the Importance of Nanoparticle Delivery for Effective Tumor PD-L1 Downregulation. Frontiers in Oncology, 10, 614365.

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Silencing Chk-α and PD-L1 with siRNA

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All siRNA were purchased from Dharmacon (Lafayette, CO, USA). Untreated cells and cells treated with nontargeted scrambled siRNA (Dharmacon, Catalog Item D-001810-10-20) or luciferase siRNA (Dharmacon, Catalog Item P-002099-01-50) were used as controls. Isoform-specific siRNAs were custom designed using Thermo Scientific siRNA Design Center (Thermo Scientific, Rockford, IL, USA). siRNA specific sequences were 5′-CAUGCUGUUCCAGUGCUCC-3′ for Chk-α, 5′-GAGGAAGACCUGAAGGUUCAGCAUA-3′ for PD-L1 #1, and 5′-CCUACUGGCAUUUGCUGAACGCAUU-3′ for PD-L1 #2.
Cells at 40 to 50% confluency were transfected with 100 nM of scrambled or luciferase siRNA, and with 50 nM or 100 nM of Chk-α- or PD-L1-specific siRNA for individual treatments. For combination siRNA treatments, 50 nM of each specific siRNA was used. Cells were treated with siRNA for 48 h because this incubation period resulted in the most effective downregulation of the target genes. D-FECT 4 (Dharmacon, Catalog Item T-2004-03) was used as the transfection agent for MDA-MB-231, Pa09C and Pa20C cells, and Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA, Catalog Item 11668019) for SUM-149 cells. All transfections were carried out based on established protocols [34 (link)].

Pacheco-Torres J., Penet M.F., Mironchik Y., Krishnamachary B, & Bhujwalla Z.M. (2021). The PD-L1 metabolic interactome intersects with choline metabolism and inflammation. Cancer & Metabolism, 9, 10.

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Chkα siRNA PEG-PEI NPs Efficiency

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The in vitro efficiency of RNA interference by Chkα siRNA PEG-PEI NPs was evaluated in 231 WT and 231 VEGF cells. All siRNA used in the study were obtained from Dharmacon™ (Lafayette, CO, USA). A previously validated Chkα siRNA sequence [25 (link)], 5′-CAUGCUGUUCCAGUGCUCC-3′, was designed using the Thermo Scientific siRNA Design Center (Thermo Scientific, Rockford, IL, USA). Approximately 0.4 × 10⁶ cells were seeded in 60 mm culture dishes and cultured overnight. The following day, 0.2 nmol of Chkα siRNA was mixed with PEG-PEI at an N/P ratio of 15 in 50 µL of Opti-MEM^TM reduced serum medium for 30 min before being added to the petri dish. The siRNA concentration was 100 nM. Untreated cells served as a negative control. Cells treated with an identical amount of Chkα siRNA mixed with DharmaFECT 4 as the transfection reagent were used as a positive control. After 24 h of incubation, cells were collected and the Chkα mRNA level of each group evaluated by RT-PCR.

Tan S., Chen Z., Mironchik Y., Mori N., Penet M.F., Si G., Krishnamachary B, & Bhujwalla Z.M. (2022). VEGF Overexpression Significantly Increases Nanoparticle-Mediated siRNA Delivery and Target-Gene Downregulation. Pharmaceutics, 14(6), 1260.

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Choline and Ethanolamine Modulate Cancer Cell Lines

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MDA-MB-231 breast cancer cells were cultured in RPMI-1640 medium containing 21 μM choline supplemented with 10% FBS and 50μm ethanolamine. Nonmalignant MCF-12A human mammary epithelial cells were grown in DMEM-Ham’s F12 medium containing 64 μM choline further supplemented with 50 μm ethanolamine. Panc-1, Pa02C, and Pa04C human pancreatic cancer cells were cultured in DMEM containing 28 μm choline and 50 μm ethanolamine. For comparison, we used human pancreatic nestin-expressing (HPNE) cells from ATCC (ATCC, Manassas, VA). HPNE cells are non-neoplastic human pancreatic cells retrovirally transduced with the human telomerase reverse transcriptase (hTERT) gene to stably express hTERT. HPNE cells were cultured according to the manufacturer’s protocol with medium that contained 28 μM choline, supplemented with 50 μm ethanolamine. Isoform-specific siRNAs were custom designed using Thermo Scientific siRNA design center (Thermo Scientific, Rockford, IL). Accession numbers NM_001277.2 for Chk-α, NM_005198.4 for Chk-β, NM_018638 for Etnk-1, and NM_018208.3 for Etnk-2 were used to design specific siRNA. While 50 nM siRNA was used in individual siRNA treatments, for combined siRNA treatment 50 nM of each specific siRNA was used. Cells were transfected with siRNA for 24 h and cell extraction was performed 48 h post siRNA treatment.

Shah T., Krishnamachary B., Wildes F., Wijnen J.P., Glunde K, & Bhujwalla Z.M. (2018). Molecular Causes of Elevated Phosphoethanolamine in Breast and Pancreatic Cancer Cells. NMR in biomedicine, 31(8), e3936.

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