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On targetplus

Manufactured by Thermo Fisher Scientific
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ON-TARGETplus is a gene silencing product from Thermo Fisher Scientific. It is designed to provide efficient and specific gene knockdown in cell-based assays.

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Lab products found in correlation

Lipofectamine rnaimax, by Thermo Fisher Scientific (8 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Sigenome, by Thermo Fisher Scientific (2 mentions) Actin, by Cell Signaling Technology (2 mentions) Smartpool, by Thermo Fisher Scientific (2 mentions) Sigenome smartpool sirna, by Thermo Fisher Scientific (1 mentions) Flexitube genesolution, by Qiagen (1 mentions) Flexitube sirna, by Qiagen (1 mentions) On targetplus non targeting sirna 1, by Thermo Fisher Scientific (1 mentions) Dharmafect 1, by Thermo Fisher Scientific (1 mentions) Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions) Synthetic oligonucleotides, by Thermo Fisher Scientific (1 mentions) Dharmafect 1 transfection reagent, by Thermo Fisher Scientific (1 mentions) Non targeting sirna, by Thermo Fisher Scientific (1 mentions) On targetplus sirna, by Thermo Fisher Scientific (1 mentions) Dharmafect, by Thermo Fisher Scientific (1 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions) Silencer select pre designed, by Horizon Discovery (1 mentions) Opti mem medium, by Thermo Fisher Scientific (1 mentions) Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)

23 protocols using on targetplus

1

Transient Transfection of SUV39H1 and BRCA1 siRNA

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For transient transfection, human SUV39H1 siRNA was purchased from Thermo Scientific (SMARTpool, ON-TARGETplus), and the SUV39H1 target sequences were CUAAGAAGCGGGUCCGUAU (J-009604-07), GGUGAAAUGGCGUGGAUAU (J-009604-08), UCGAGUACCUGUGCGAUUA (J-009604-09), and CAAAUCGUGUGGUACAGAA (J-009604-10). Human BRCA1 siRNA was purchased from Thermo Scientific (SMARTpool, ON-TARGETplus), and the BRCA1 target sequence was CAACAUGCCCACAGAUCAA (J-003461-09), CCAAAGCGAGCAAGAGAAU (J-003461-10), UGAUAAAGCUCCAGCAGGA (J-003461-11), and GAAGGAGCUUUCAUCAUUC (J-003461-12). All siRNAs were transfected with oligofectamine for 48-72 hours for further analysis.
Mo W., Liu Q., Lin C.C., Dai H., Peng Y., Liang Y., Peng G., Meric-Bernstam F., Mills G.B., Li K, & Lin S.Y. (2015). mTOR inhibitors suppress homologous recombination repair and synergize with PARP inhibitors via regulating SUV39H1 in BRCA-proficient triple-negative breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(7), 1699-1712.
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2

RNAi Experiments for Targeted Silencing

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Primary RNAi experiments were performed using siGENOME SMARTpool siRNA (Thermo Scientific). Secondary validation of silencing phenotypes was conducted with individual siRNA oligonucleotides of siGENOME or ON-TARGETplus design (Thermo Scientific), or pooled siRNA from FlexiTube GeneSolution (QIAGEN). Details of individual siRNA sequences as well as non-targeting/negative control siRNA used for normalization and statistical comparisons are provided as supplementary information (tables S6 and S7). Transfections were carried out with Lipofectamine RNAiMAX according to manufacturer’s instructions. Cells were transfected with siRNA at a final concentration of 10 nM (siGENOME), 15 nM (QIAGEN) or 25 nM (ON-TARGETplus) 72 hours prior to assaying the impact of silencing.
Locard-Paulet M., Lim L., Veluscek G., McMahon K., Sinclair J., van Weverwijk A., Worboys J.D., Yuan Y., Isacke C.M, & Jørgensen C. (2016). Phosphoproteomic analysis of tumor-endothelial signaling identifies EPHA2 as a negative regulator of transendothelial migration. Science signaling, 9(414), ra15.
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3

Silencing Rer1, Hrd1, and Calnexin in HeLa Cells

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In human Rer1, siRNA Rer1 #1 (FlexiTube siRNA; Qiagen) and #2 (ON-TARGETplus; Thermo Fisher Scientific) target the mRNA 5′-noncoding and coding region from -13 to 8 (5′-UGCGAGUUACAGAAUGUCUGA-3′) and the coding region from 285 to 303 (5′-AGAUGACGGUCCUUCGCUA-3′), respectively, relative to the translation initiation site. siRNA Hrd1 and calnexin (FlexiTube siRNA; Qiagen) target the human Hrd1 mRNA coding region from 404 to 422 (5′-GGUGAUGGGCAAGGUGUUC-3′) and the human calnexin mRNA 3′-noncoding region from 1914 to 1934 (5′-ACACUAGUCUGUGUAACUUUA-3′), respectively, relative to the initiation site. AllStars Negative Control siRNA (Qiagen) and ON-TARGETplus Non-targeting siRNA #1 (Thermo Fisher Scientific) were used as control siRNAs. siRNAs were transfected into HeLa cells with Lipofectamine RNAiMAX (Thermo Fisher Scientific), according to the manufacturer's instructions. After 72 h of transfection, the cells were used in subsequent experiments.
Yamasaki A., Hara T., Maejima I., Sato M., Sato K, & Sato K. (2014). Rer1p regulates the ER retention of immature rhodopsin and modulates its intracellular trafficking. Scientific Reports, 4, 5973.
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4

PDE3A Knockdown and Compound 3 Assay

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HeLa cells were plated in 96-well plates and transfected after 24 hours with PDE3A and Non-Targeting siRNA smartpools (On Target Plus, Thermo Scientific) according to the manufacturers recommendations. HeLa cell lysate was obtained 24 hours and 72 hours after transfection and immunoblotted for PDE3A and Actin (1:20,000, Cell Signaling) as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting. HeLa cells were treated for 48 hours with indicated concentrations of Compound 3 (supplementary figure 2). Cell viability was assessed as described in Compound library screening in NCI-H1734 and A549 cell lines.
de Waal L., Lewis T.A., Rees M.G., Tsherniak A., Wu X., Choi P.S., Gechijian L., Hartigan C., Faloon P.W., Hickey M.J., Tolliday N., Carr S.A., Clemons P.A., Munoz B., Wagner B.K., Shamji A.F., Koehler A.N., Schenone M., Burgin A.B., Schreiber S.L., Greulich H, & Meyerson M. (2015). Identification of cancer cytotoxic modulators of PDE3A by predictive chemogenomics. Nature chemical biology, 12(2), 102-108.
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5

Knockdown of AMPK Subunits in Cells

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siRNAs were synthesized and constructed according to the manufacturer’s protocol (siGENOME and ON-TARGETplus; Thermo Fisher Scientific., Lafayette, CO, USA.). The following targeting sequences were used: ON-TARGETplus SMARTpool for rat AMPKα1 (catalog number: L-091373-00-0010) and rat AMPKα2 (catalog number: L-100623-00-0010). Transfection agent with a scrambled siRNA (Dharmacon RNAi control; Thermo Fisher Scientific) served as a control. Cells were seeded into 6-well plates at a concentration of 1×105 cells/well and incubated for 24 h before transfection. Cells were transfected with siRNAs in serum-free DMEM using DharmaFECT1 (Thermo Fisher Scientific) according to the manufacturer’s protocol. After 24 h of incubation, the transfected cells were serum-deprived for 8 h, and then used in the following experiments.
Chen K.H., Hsu H.H., Lee C.C., Yen T.H., Ko Y.C., Yang C.W, & Hung C.C. (2014). The AMPK Agonist AICAR Inhibits TGF-β1 Induced Activation of Kidney Myofibroblasts. PLoS ONE, 9(9), e106554.
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6

MCF-7 Cell siRNA Transfection Protocol

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Pool 1 SRA siRNA (SRA Smartpool, Dharmacon, D-120329) has been described [29 (link)]. Pool 1 nontarget siRNA was designed by scrambling the siRNA sequences in pool 1 SRA siRNA. Sequences of these siRNAs are listed in Table S1. Pool 2 SRA siRNA is ON-TARGETplus siRNA pool for human SRA1 (Thermo Scientific, L-027192-00-0005 and LU-027192-00-0002). Pool 2 Control siRNA is ON-TARGETplus Non-targeting control pool (Thermo Scientific, D-001810-10-05).
For siRNA transfection, corresponding siRNAs were reverse-transfected into 200,000-300,000 MCF-7 cells with Lipofectamine® RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions.
McKay D.B., Xi L., Barthel K.K, & Cech T.R. (2014). Structure and Function of Steroid Receptor RNA Activator Protein, the Proposed Partner of SRA Non-coding RNA. Journal of molecular biology, 426(8), 1766-1785.
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7

RNA Interference Targeting Mitotic Proteins

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The synthetic oligonucleotides targeting human Hec1 and CENP-E for RNAi were obtained from Invitrogen (Stealth). The sequences were as follows; Hec1 (5′-UCAGCCAUUCUUGACCAGAAAUUAA-3′), and CENP-E (5′-CGGCUCAAGGAAGGCUGUAAUAUAA-3′). The siRNA sequences targeting for Zw10, Spindly and Kid were obtained from JBioS. The sequences were as follows: Zw10 (5′-UGAUCAAUGUGCUGUUCAATT-3′), Spindly (5′-GAAAGGGUCUCAAACUGAATT-3′), and Kid (5′-AAGAUUGGAGCUACUCGUCGUTT-3′). The sequences of mixed siRNAs targeting for Ska3, obtained from ThermoFisher Scientific (ON-TARGETplus), were as follows: 5′-GGAAGAGCCCGUAAUUGUA-3′, 5′-GAUCGUACUUCGUUGGUUU-3′, 5′-AAUCCAGGCUCAAUGAUAA-3′, and 5′-CAUCGUAUCCCAAGUUCUA-3′.
Itoh G., Ikeda M., Iemura K., Amin M.A., Kuriyama S., Tanaka M., Mizuno N., Osakada H., Haraguchi T, & Tanaka K. (2018). Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment. Scientific Reports, 8, 3888.
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8

siRNA Knockdown in Neuronal Cultures

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Primary neuron or neuroblastoma cultures were treated with 25nM siRNA SMARTpools against target genes or a nontargeting control pool (ON-TARGETplus, ThermoScientific). siRNA pools were delivered in a 1:500 dilution of DharmaFect 1 transfection reagent (ThermoScientific) in antibiotic-free culture medium for 48 hours. Following siRNA treatment, cells were rinsed and returned to normal culture medium for further experimentation.
Daniels B.P., Kofman S.B., Smith J.R., Norris G.T., Snyder A.G., Kolb J.P., Gao X., Locasale J.W., Martinez J., Gale M J.r., Loo Y.M, & Oberst A. (2019). The nucleotide sensor ZBP1 and kinase RIPK3 induce the enzyme IRG1 to promote an antiviral metabolic state in neurons. Immunity, 50(1), 64-76.e4.
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9

siRNA Transfection and TGF-β1 Treatment

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Small inhibitory RNAs (siRNAs) were obtained from Thermo Scientific (ON-TARGET plus SMART human SDC4 and ON-TARGET plus Non-Targeting siRNA, Waltham, MA, USA). The transfection of siRNAs was performed according to the manufacturer’s protocol. The WI-38 cells were incubated in growth medium for 24 h, and a final concentration of 100 nM siRNA was added to the cells. Lipofectamine RNAiMAX (Invitrogen) was used as a transfection medium. After 24 h, the cells were washed and incubated with or without TGF-β1 for specified times.
Tanino Y., Wang X., Nikaido T., Misa K., Sato Y., Togawa R., Kawamata T., Kikuchi M., Frevert C.W., Tanino M., Kojima T, & Shibata Y. (2019). Syndecan-4 Inhibits the Development of Pulmonary Fibrosis by Attenuating TGF-β Signaling. International Journal of Molecular Sciences, 20(20), 4989.
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10

IFITM3 Knockdown in ORL Cell Lines

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Synthetic oligonucleotides ON-TARGET plus siRNA AUGGAUAGAUCAGGAGGCA (si18, J-014116-18) and TGCTGATCTTCCAGGCCTA (si19, J-014116-19) targeting human IFITM3, and ON-TARGET plus Non-Targeting (NT) siRNA UGGUUUACAUGUCGACUAA were purchased from Thermo Scientific (MA, USA). Two ORL cell lines (ORL-150 and ORL-204) that were found to overexpress IFITM3 were used for the knock-down experiments (Supplementary Fig. 1a). Cells were seeded at a density of 2 × 105/well in a 6-well plate and incubated for 24 h prior to transfection. Cells were transfected with 50 nM siRNA using cationic lipid DharmaFECT (Thermo Scientific, MA, USA) and incubated for an additional 48 h prior to harvesting for analysis. The level of IFITM3 expression after siRNA knockdown was determined by qRT-PCR and Western blotting.
Gan C.P., Sam K.K., Yee P.S., Zainal N.S., Lee B.K., Rahman Z.A., Patel V., Tan A.C., Zain R.B, & Cheong S.C. (2019). IFITM3 knockdown reduces the expression of CCND1 and CDK4 and suppresses the growth of oral squamous cell carcinoma cells. Cellular oncology (Dordrecht), 42(4), 477-490.
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