On target plus non targeting control sirna 1

Manufactured by Horizon Discovery

Sourced in United States

ON-TARGET plus Non-targeting Control siRNA #1 is a laboratory reagent designed for use as a control in RNA interference (RNAi) experiments. It is a double-stranded RNA molecule that does not target any known mammalian gene, allowing for the evaluation of off-target effects in RNAi studies.

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Lab products found in correlation

Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions) Dharmafect 4, by Horizon Discovery (1 mentions) Siscr, by Horizon Discovery (1 mentions) On targetplus smartpool, by Horizon Discovery (1 mentions) Hela tet on, by Takara Bio (1 mentions) Hek293t, by American Type Culture Collection (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Hek293, by American Type Culture Collection (1 mentions) Scanr microscope, by Olympus (1 mentions)

5 protocols using on target plus non targeting control sirna 1

Dissecting YAP's Role in C2C12 Cells

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C2C12 cells were seeded at a density of 5*10⁴ cells/well. Twenty-four hours later, cells were transfected with either siRNA targeting YAP (SMARTpool ON-TARGETplus YAP1, Dharmacon, Lafayette, CO, USA), or scrambled control siRNA (ON-TARGET plus Non-targeting Control siRNA #1, Dharmacon, Lafayette, CO, USA), from now on referred to as siYAP and siScr, respectively, at a final concentration of 50 nM per well. The siRNAs were mixed with 2.5 μl Lipofectamine RNAiMAX (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) transfection reagent per well. The next day the cells were starved for 3 h, stimulated for 5 min with FLIPUS, and analyzed by BrdU-ELISA as described below.
Additionally, several genes were selected as potential YAP targets (see the primers’ list below) and their expression was quantified by qRT-PCR in both siYAP and siScr samples.

Puts R., Rikeit P., Ruschke K., Knaus P., Schreivogel S, & Raum K. (2018). Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound (FLIPUS) enhances proliferation of murine mesenchymal precursors. PLoS ONE, 13(10), e0206041.

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Transient Silencing of NME1 and NME2 Genes

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Transient transfection of cells with small interfering RNA molecules (siRNA) was used for silencing the NME1 and NME2 genes. By direct silencing in adherent cells, we performed transfection with two siRNAs (ON-TARGETplus Human NME1 siRNA-Smartpool L-006821-00-0005 and ON-TARGETplus Human NME2 siRNA-Smartpool L-005102-00-0005, Dharmacon, Lafayette, CO, USA) using DharmaFECT 4 (Dharmacon, Lafayette, CO, USA) transfection reagent according to the manufacturer’s protocol. In parallel, we also transfected the cells with non-targeting, control siRNA (ON-TARGETplus non-targeting control siRNA#1, Dharmacon, Lafayette, CO, USA).
To determine the efficacy of silencing by western blot, cells were harvested 48 h after transfection. A portion of the cells was reseeded and collected 96 h later to verify the persistence of the silencing over a longer period required for the MTT assay.

Radić M., Vlašić I., Jazvinšćak Jembrek M., Horvat A., Tadijan A., Sabol M., Dužević M., Herak Bosnar M, & Slade N. (2022). Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance. International Journal of Molecular Sciences, 23(17), 9910.

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Investigating miR-181c Regulation in Cardiac Cells

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miR‐181c overexpression was performed by transient transfection using either scramble (Scr, Qiagen) or miR‐181c mimic (Cat# MIMAT0000674, Qiagen) in H9c2 or NMVMs. In vitro knocking down of Sp1 or MICU1 expression was performed using small interference RNAs against Sp1 (siSp1; ON‐TARGETplus SMART Pool Cat# L‐040633‐02‐0005, Dharmacon, Lafayette, CO) and MICU1 (siMICU1; ON‐TARGETplus SMART Pool Cat# L‐053388‐01, Dharmacon, Lafayette, CO). Both siSp1 and siMICU1 transfected groups were compared with scramble (siScr, ON‐TARGETplus Non‐targeting Control siRNA #1, Cat# D‐001810‐01‐05, Dharmacon, Lafayette, CO) transfected group. All transfections were carried out using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA).

Banavath H.N., Roman B., Mackowski N., Biswas D., Afzal J., Nomura Y., Solhjoo S., O'Rourke B., Kohr M., Murphy E., Steenbergen C, & Das S. (2019). miR‐181c Activates Mitochondrial Calcium Uptake by Regulating MICU1 in the Heart. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(24), e012919.

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Cell Line Maintenance and Transfection Protocols

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HeLa ^Tet-on (Clontech), HEK293 (American Type Culture Collection, ATCC), HEK293T (ATCC), and T-REx293 IRE1-GFP cells (Li et al., 2010 (link)) were maintained at 37C, 5% CO2 in Dulbecco’s Modified Eagle’s Medium (Sigma) supplemented with 4500 mg/L glucose, L-glutamine, sodium pyruvate, sodium bicarbonate, 10% fetal bovine serum (Sigma), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma). T-REx293 IRE1-GFP cells were kindly provided by Dr. Peter Walter (UCSF/HHMI). Transient transfection of plasmids were performed using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Transient gene knockdown was accomplished by transfection of small double-stranded interfering RNAs (siRNA) into cells using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The siRNAs were synthesized by Dharmacon using the sequence information in the Key resources table. Silencer Negative Control No. 1 siRNA (Ambion) or ON-TARGET plus Non-targeting Control siRNA #1 (Dharmacon) were used as vehicle negative controls.

Wu I.H., Yoon J.S., Yang Q., Liu Y., Skach W, & Thomas P. (2021). A role for the ribosome-associated complex in activation of the IRE1 branch of UPR. Cell reports, 35(10), 109217.

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Systematic siRNA Screening of Ubiquitin Pathway

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siRNA screening was conducted using the RTF SMARTpool siRNA Library - Human Ubiquitin Conjugation Subset 1 (Dharmacon), consisting of two siRNA-coated plates (H-105615, Lot 12115). Plates were used to reverse-transfect cells at 50 nM final siRNA concentration into optical imaging plates. 72 hr after transfection, eYFP-CRAF degradation was initiated by addition of AUY922 for 8 hr. After incubation, cells were washed and fixed for 10 min using 4% paraformaldehyde in PBS, permeabilized using 0.3% Triton in PBS, and stained with 0.5 μg/mL DAPI in PBS for 15 min. After final wash, all cell samples were immediately imaged using the Olympus ScanR microscope at 10× magnification. Images were subsequently analyzed using ScanR Analysis proprietary software. At minimum 10,000 cells were imaged per individual sample, per experiment.
siRNA verification experiments were conducted in a similar format using specific siRNAs (Table S2). siRNA controls used ON-TARGETplus Non-Targeting Control siRNA #1 (Dharmacon).

Li Z., Zhou L., Prodromou C., Savic V, & Pearl L.H. (2017). HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients. Cell Reports, 19(12), 2515-2528.

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