Sirna

Manufactured by Horizon Discovery

Sourced in United States, United Kingdom, Germany, Netherlands

SiRNA is a laboratory tool used for gene silencing. It functions by targeting and degrading specific messenger RNA (mRNA) molecules, thereby reducing the expression of the corresponding gene. SiRNA technology enables researchers to study the effects of gene knockdown on cellular processes and pathways.

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Lab products found in correlation

459 protocols using sirna

Silencing of Key Signaling Proteins

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Transient silencing was obtained by transfection of siRNA (Sigma Genosys or Life Technologies). Briefly were plated on matrigel coated coverlips to 30–50% confluence the day before transfection and transfected using lipofectamine 2000 (Life Technologies) according to manufacturer’s instructions. The day after transfection cells were serum deprived for further 18 hours before immunofluorescences or western blotting.
Validated siRNA DGKα [17] (link) sense 5′ GGAUGGCGAGAUGGCUAAAtt 3′ antisense 5′UUUAGCCAUCUCGCCAUCCgg 3′.
siRNA PKCζ sense 5′CGUUCGACAUCAUCACCGAtt3′antisense 5′UCGGUGAUGAUGUCGAACGgg3′.
siRNA PKCι sense 5′CGUUCGACAUCAUCACCGAtt3′ antisense 5′UCGGUGAUGAUGUCGAACGgg3′.
siRNA β1 integrin sense 5′GGAGGAAUGUUACACGGCU3′ antisense 5′ AGCCGUGUAACAUUCCUCCag 3′.
siRNA RCP: ON-TARGETplus RAB11FIP1 siRNA L-015968-00-0005 (Dharmacon). Silencer negative control siRNA AM4611 (Life Technologies) was used as negative control.

Rainero E., Cianflone C., Porporato P.E., Chianale F., Malacarne V., Bettio V., Ruffo E., Ferrara M., Benecchia F., Capello D., Paster W., Locatelli I., Bertoni A., Filigheddu N., Sinigaglia F., Norman J.C., Baldanzi G, & Graziani A. (2014). The Diacylglycerol Kinase α/Atypical PKC/β1 Integrin Pathway in SDF-1α Mammary Carcinoma Invasiveness. PLoS ONE, 9(6), e97144.

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Targeted gene knockdown using siRNA

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Scrambled siRNA (5′-GCAGUGUCUCCACGUACUAdTdT-3′), siRNAs against FDXR (siFDXR#1:5′-GCTCAGCAGCATTGGG-TAT-3′ and #2: 5′-GCTCAGCAGCATTGGGTAT-3′), siRNA against human p53 (5′-CACCUUGAUCCAGCGGACUUAdTdT-3′) and siRNA against human IRP2 (5′-GCGAUUUC-CAGGCUUGCUUdTdT-3′) were purchased from Dharmacon (Chicago, IL, USA). For siRNA transfection, siRNAMax Lipid Reagent (Thermo Fisher Scientific) was used according to the user’s manual.

Zhang Y., Feng X., Zhang J., Chen M., Huang E, & Chen X. (2019). Iron regulatory protein 2 is a suppressor of mutant p53 in tumorigenesis. Oncogene, 38(35), 6256-6269.

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Silencing of Iron-Sulfur Cluster Proteins

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Scrambled siRNA (5′- GGC CGA UUG UCA AAU AAU U- 3′), siRNAs against FDXR (5′-CAC CUU GAU CCA GCG GAC UUA -3′ and 5′- GCU CAG CAG CAU UGG GUA U -3′), siRNAs against FDX2 (5′- GCU GCA AUA AAU CGA UAA CAC -3′ and 5′- GCU GCC AGA UUG UCU GAC AC -3′) and siRNA against IRP2 (5′- GCA AAC AUG UGU CCG GAA U -3′) were purchased from Dharmacon (Chicago, IL, USA). RNAiMax from Life Technology (Carlsbad, CA, USA) was used for siRNA transfection according to the user manual. Cells were transfected with scrambled or target siRNA at 25 nM for 3 days.

Zhang J., Kong X., Zhang Y., Sun W., Wang J., Chen M, & Chen X. (2020). FDXR regulates TP73 tumor suppressor via IRP2 to modulate aging and tumor suppression. The Journal of pathology, 251(3), 284-296.

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PREP1 Silencing in HeLa and BJ Cells

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To down-regulate PREP1 in HeLa cells and BJ fibroblasts a mixture of two independent siRNA (Dharmacon, Lafayette, USA) were used (siRNA 607 = GATTTCTGCAGTCGATACA; siRNA 900 = CTCCCAGCTTCAGTTACAG). As control we used an siRNA targeting firely luciferase (CATCACGTACGCGGAATAC). 10 nM dsRNA were transfected using RNAi Max lipofectamine (Invitrogen). To assess PREP1 down-regulation, 30 µg protein extracts were subjected to SDS-PAGE gel and PREP1 was detected using a monoclonal CH12.2 antibody.

Palmigiano A., Santaniello F., Cerutti A., Penkov D., Purushothaman D., Makhija E., Luzi L., di Fagagna F.D., Pelicci P.G., Shivashankar V., Dellino G.I, & Blasi F. (2018). PREP1 tumor suppressor protects the late-replicating DNA by controlling its replication timing and symmetry. Scientific Reports, 8, 3198.

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Silencing Proteins with siRNA Transfection

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Small interfering RNAs (siRNAs; Dharmacon Inc.) were transfected using Lipofectamine RNAi MAX (Invitrogen) as per the manufacturer’s protocol. The final concentration of the siRNA was 40 nM and the cells were lysed 72 h post-transfection. For SET7 siRNA, SMARTpool were purchased from Dharmacon Inc. For FEN1, the sequence of the siRNA used was 5′-GUU CUC UGA GGA GCG AAU C-3′. Luciferase siRNA was used as control.

Thandapani P., Couturier A.M., Yu Z., Li X., Couture J.F., Li S., Masson J.Y, & Richard S. (2017). Lysine methylation of FEN1 by SET7 is essential for its cellular response to replicative stress. Oncotarget, 8(39), 64918-64931.

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siRNA-Mediated Knockdown Experiment

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Scrambled siRNA (5′-GCA GUG UCU CCA CGU ACU AdTdT-3′), siRNAs against IRP2 (siIRP2#1:5′-GCG AUU UCC AGG CUU GCU UdTdT −3′ and #2: 5′-GCA AAC AUG UGU CCG GAA dTdT-3′), and siRNA against human TAp63 (5′-GAU GGU GCG ACA AAC AAG AdTdT-3′) were purchased from Dharmacon. For siRNA transfection, RNAiMax Lipid Reagent (Thermo Fisher Scientific) was used according to the manufacturer’s manual. The siRNAs were transfected into the cells at the final concentration of 30 nmol/L for 3 days.

Zhang Y., Feng X., Zhang J, & Chen X. (2020). Iron Regulatory Protein 2 Exerts its Oncogenic Activities by Suppressing TAp63 Expression. Molecular cancer research : MCR, 18(7), 1039-1049.

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Knockdown of LEF-1 in Cell Transfection

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Cells were transfected using Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific, MA, USA), according to the manufacturer’s protocol, with small interfering RNAs (siRNAs, final concentration 50 nM; Dharmacon, Horizon Discovery, Cambridge, UK) targeting LEF-1, or a negative control, for which four different siRNA sequences were pooled.^{44 (link)} Cells were incubated overnight at 37 °C before medium changing. Samples were prepared 72 h after transfection.

Dixon S.W., Collard T.J., Mortensson E.M., Legge D.N., Chambers A.C., Greenhough A., Creed T.J, & Williams A.C. (2021). 5-Aminosalicylic acid inhibits stem cell function in human adenoma-derived cells: implications for chemoprophylaxis in colorectal tumorigenesis. British Journal of Cancer, 124(12), 1959-1969.

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Plasmid and siRNA Protocols for FOXM1B and Met

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The plasmid pcDNA3.1-FOXM1B (pFOXM1) and control vector plasmid pcDNA3.1 were described previously.^{24 (link)} Small interfering RNAs (siRNAs) targeting Met was synthesized by Dharmacon, and the sequence of siRNA#1 5'-GTGCAGTATCCTCTGACAG-3' and siRNA#2 5’-AAGTGCAGTATCCTCTGACAG-3’ were reported previously.^{25 (link),26 (link)} SiRNA targeting FOXM1 (siFOXM1) was described previously.²⁷

Cui J., Xia T., Xie D., Gao Y., Jia Z., Wei D., Wang L., Huang S., Quan M, & Xie K. (2016). HGF/Met and FOXM1 Form a Positive Feedback Loop and Render Pancreatic Cancer Cells Resistance to Met Inhibition and Aggressive Phenotypes. Oncogene, 35(36), 4708-4718.

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siRNA Transfection and Protein Knockdown

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Small interfering RNAs (siRNAs; Dharmacon Inc.) were transfected using Lipofectamine RNAi MAX (Invitrogen, Carlsbad, CA) as per the manufacturer's protocol. The final concentration of the siRNA was 40 nM and the cells were lysed 72 hr post-transfection. The siRNA target sequence for PRMT1 was 5′-CGU CAA AGC CAA CAA GUU A-3′. The siRNA target sequences for Aven were siAven 5′-GAG GAG AAA GAA UGG GAU AUU-3′. For SMN, TDRD3, and DHX36 siRNAs, SMARTpools were purchased from Dharmacon Inc.

Thandapani P., Song J., Gandin V., Cai Y., Rouleau S.G., Garant J.M., Boisvert F.M., Yu Z., Perreault J.P., Topisirovic I, & Richard S. (2015). Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes. eLife, 4, e06234.

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Characterization of CHD2 and CSTF3 Regulators

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A final concentration of 100–120 nmol/L siRNA was used, and siRNAs for siCHD2 (D-060656-03) and siCSTF3 (D-051652-17) were purchased from Dharmacon. The cDNA sequence of CSTF3 with a His tag sequence was cloned into the pCD513B-1 plasmid to generate the CSTF3-His plasmid. CHD2¹⁻⁴⁵⁵, CHD2^456−955 or CHD2^955−1827 was inserted into the pEGFP-C2 plasmid to generate CHD2-A, CHD2-B and CHD2-C plasmids. The CHD2^456−955 fragment was cloned into the pLVX-IRES-Puro-GFP lentivirus plasmid to generate the pLVX-CHD2-B plasmid. The +933 to +1244 part of the Oct4 (NM_013633.3) cDNA sequence was cloned into the pEGFP-C2 plasmid to generate Oct4 wt and Oct4 mut. The primers used in the above experiments are presented in Table S2. Transient transfection was performed with TurboFect Transfection Reagent (Thermo Fisher Scientific, Inc. R0531) according to the manufacturer’s protocol. HEK293T cells were transfected with pLVX-CHD2-B, psPAX2 and pMD2.G plasmids at a 4:3:1 ratio to package lentivirus pLVX-CHD2-B.

Min Z., Xin H., Liu X., Wan J., Fan Z., Rao X., Fan J., Yang L, & Li D. (2022). Chromodomain helicase DNA-binding domain 2 maintains spermatogonial self-renewal by promoting chromatin accessibility and mRNA stability. iScience, 25(12), 105552.

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