Silencer Select siRNA oligonucleotides specific to CP110 (#1 sense, 5′-GCAAAACCAGAAUACGAGATT-3′, and antisense, 5′-UCUCGUAUUCUGGUUUUGCAT-3′; and #2 sense, 5′-CAAGCGGACUCACUCCAUATT-3′, and antisense, 5′-UAUGGAGUGAGUCCGCUUGAG-3′) or GAPDH as an experimental control (sense, 5′-UGGUUUACAUGUUCCAAUATT-3′, and antisense, 5′-UAUUGGAACAUGUAAACCATG-3′) were purchased from Ambion. For suspension cells, 50 nmol siRNA was transfected using Amaxa Nucleofector Kit V (Jurkat) or Kit T (NALM-6) according to the manufacturer’s instructions (Lonza). Cells were analyzed by Western blotting and immunofluorescence microscopy 72 h (Jurkat) or 100 h (NALM-6) after transfection. For hTERT-RPE1 cells, 50 nmol siRNA was complexed with Oligofectamine (Invitrogen) in serum-free OptiMEM (Gibco) according to the manufacturer’s instructions. 24 h after transfection, cells were serum starved as described for 24 h before analysis by Western blotting and immunofluorescence microscopy.
Silencer select sirna oligonucleotides
Manufactured by Thermo Fisher Scientific
Sourced in United States
Silencer Select siRNA oligonucleotides are designed for gene silencing applications. They are synthetic, chemically modified short interfering RNA (siRNA) molecules that target and degrade specific mRNA transcripts, thereby reducing the expression of target genes.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (5 mentions)
Oligofectamine, by Thermo Fisher Scientific (4 mentions)
Pnfκb luciferase, by BD (1 mentions)
Pduo md2 htlr4, by InvivoGen (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter system, by Promega (1 mentions)
On targetplus non targeting control pool, by Horizon Discovery (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
8 protocols using silencer select sirna oligonucleotides
1
Silencing CP110 and GAPDH in Cell Lines
2
Silencing TLR2 and TLR4 in Breast Cancer
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siRNA transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA): 2 μM of the following silencer select siRNA oligonucleotides from Ambion (Carlsbad, CA, USA) were used; Silencer Select Negative Control #2: 4390846, siTLR2 #1: s168, siTLR2 #2: s170, siTLR4 #1: s14194, siTLR4 #2: s14195. Analyses were performed 48 h and 72 h post transfection. For luciferase assays, breast cancer cells were co-transfected using Lipofectamine 2000 with a total of 0.6 μg pNFκB-luciferase (BD Biosciences) and 0.06 μg TK-renilla-luciferase (Promega, Madison, WI, USA) plasmids and was subsequently analyzed using Dual-Luciferase Reporter System (Promega). For TLR4 transfections breast cancer cells were transfected using Lipofectamine 2000 with a total of 1.0 μg pDUO-MD2/hTLR4 or pUNOI-hTLR4-GFP (Invivogen, San Diego, CA, USA) per 24 wells for 72 h or 48 h, respectively, and was subsequently analyzed using immunofluorescence (×40 magnification) or ELISA as described in the figure legends.
3
Knockdown of C-NAP1 and GAPDH in hTERT-RPE1 Cells
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hTERT-RPE1 cells were transfected with an ON-TARGETplus SMART pool of RNA duplexes inhibitory to C-NAP1 (L-012364-00-0005; Dharmacon), Silencer Select siRNA oligonucleotides specific to GAPDH (s5573; Ambion), or an ON-TARGETplus Non-targeting Control Pool (D-001810-10-05; Dharmacon) using Oligofectamine (Invitrogen). A 50- or 100-nmol amount of siRNA was complexed with Oligofectamine in serum-free OptiMEM (Gibco) and added to cells at 30–40% confluency. Serum was added 5 h after transfection and fresh medium 24 h after transfection. Cells were analyzed 24 and 48 h after transfection. Where indicated, cells were serum starved 24 h after transfection.
4
Silencing Gene Expression in hTERT-RPE1 Cells
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hTERT-RPE1 cells were plated to attain 30–40% confluency on the day of transfection. On the next day, 50 nmol of custom siRNA (5′-AAGGAUGGUUCUAAGCAUAUC-3′; Jeong et al., 2007 (link)) or Silencer Select siRNA oligonucleotides specific to GAPDH (s5573; Ambion) was complexed with Oligofectamine (Invitrogen) in Opti-MEM (Gibco) for 20 min before addition to cells. After 4-h incubation of cells with the Oligofectamine-siRNA complexes, medium supplemented with 30% FBS was added, and cells were incubated for 48 h. Where indicated, cells were transfected for 36 h before serum starvation for 24 h.
5
Silencing SULT1A1 Gene Expression
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Cells were seeded at 5000 cells per well in 96-well tissue culture plates and allowed to adhere overnight. Cells were transfected with 20 µM of Silencer™ Select siRNA oligonucleotides targeting human SULT1A1 (s13613, Ambion) or a non-targeting siRNA negative control (Negative Control No. 1 siRNA, Ambion). Cells were transfected using Lipofectamine RNAi max (Invitrogen) according to manufacturer’s specifications. After 24 h of transfection media was replaced with normal growth media.
6
Tollip Silencer Select siRNA Knockdown
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Silencer Select siRNA oligonucleotides 5’-GCUGUUGAUUUAAGGCACAtt-3’ (S29037; designated #1) and 5’-GACUCUUUCUAUCUCGAGAtt-3’ (S29038; designated #2) against Tollip were purchased from Ambion. Two non-specific Silencer Select siRNA oligonucleotides (#4390843 and #4390846) served as negative controls.
7
Knocking Down ESR2 Expression in Breast Cancer Cells
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To knockdown the ESR2 expression induced by the Sal treatment, MDA-MB-468 cells (5 × 104 cells/well) were seeded on a 6-well plate overnight and then simultaneously treated with Sal (0.5 μM) and transfected with Silencer Select siRNA oligonucleotides (ThermoFisher Scientific, Waltham, MA), according to the manufacturer instructions. Briefly, siRNA (60 nM) was mixed with RNAiMAX transfection reagent in the presence of OptiMEM reduced serum medium for 5 min at room temperature. The siRNA-lipid complex and Sal were then co-incubated with the cells. An equimolar mixture of 3 different pre-designed siRNA (20 nM each) targeting multiple regions of ESR2 gene (Table S1 ) was used to silence the estrogen expression. Silencer control 1 (ctl1) and control 2 (ctl2) siRNA, not able to interact with any human RNA, were used as negative controls. 72 h after siRNA transfection and drug treatment, cells were harvested prior to performing FACS analysis and cell viability assay.
8
Silencer Select siRNA Knockdown Protocol
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Cells were transfected with Silencer Select siRNA oligonucleotides (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, 5 nM siRNA was mixed with RNAiMAX transfection reagent (Thermo Fisher Scientific) in the presence of Opti-MEM (Thermo Fisher Scientific) for 5 min at room temperature (RT). The solution was transfected into osteoprogenitor cells in the presence of growth media or mononuclear cells in macrophage induction media for 2 days. The following siRNA oligonucleotides were used: ADORA2B (A2BR; catalog no. 4390771; ID, s62047), ESR1 (catalog no. 4390771; ID, s65686), ESR2 (catalog no. 4390771; ID, s65689), and negative control #1 (catalog no. 4390843). A concentration of 5 nM negative control siRNA was used for single ER knockdown, while a concentration of 10 nM negative control siRNA was used for dual ER knockdown.
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