Sigenome non targeting sirna pool 1

Manufactured by Horizon Discovery

Sourced in United States

The SiGENOME Non-Targeting siRNA Pool #1 is a collection of small interfering RNA (siRNA) molecules designed to target no known human genes. This product is intended for use as a control in RNA interference (RNAi) experiments.

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Lab products found in correlation

27 protocols using sigenome non targeting sirna pool 1

PRAME Silencing in MDA-MB-468 Cells

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The methodology for PRAME overexpression in the MDA‐MB‐468 cell line has been described previously.¹¹ Transient silencing of PRAME was conducted using a pool of PRAME‐specific siRNA (siGENOME SMARTpool, M‐012188‐00‐0010, Dharmacon) or scrambled control siRNA (siGENOME Non‐Targeting siRNA Pool#1, D‐001206‐13‐20, Dharmacon) and DharmaFect transfection reagent (Dharmacon), according to manufacturer's guidelines.

Naik A., Thomas R., Al‐Khadairi G., Bacha R., Hendrickx W, & Decock J. (2021). Cancer testis antigen PRAME: An anti‐cancer target with immunomodulatory potential. Journal of Cellular and Molecular Medicine, 25(22), 10376-10388.

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Silencing Bcl-xL Expression Using siRNA

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PAGE purified siRNAs were synthesized and annealed by the Eurogentec Company (Liège, Belgium). Specific double-stranded 21 nt RNA oligonucleotides forming a 19 bp duplex core with 2 nt 3′ overhangs were used to silence Bcl-x_L expression (5′-auuggugagucggaucgca-3′, noted siXL). siGENOME Non-Targeting siRNA Pool#1 (noted siCT) was purchased from Dharmacon. siRNA duplexes were transfected using the INTERFERin™ transfection reagent according to the manufacturer’s instructions (Polyplus-Transfection, France). Briefly, cells were seeded in 25 cm² flasks to reach 30–50 % of confluence at the time of transfection. The transfection reagent and the siRNAs were mixed and complex formation was allowed to proceed for 15 min at room temperature before to be applied to cells. After indicated time, cells were trypsinized and washed with ice-cold PBS 1X before to be analyzed.

Bonnefond M.L., Lambert B., Giffard F., Abeilard E., Brotin E., Louis M.H., Gueye M.S., Gauduchon P., Poulain L, & N’Diaye M. (2015). Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation. Apoptosis, 20(4), 535-550.

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BCL2 Knockdown Viability Assay

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Cells were plated 24 hours before transfection in 6-well or 96-well plates. Cells were transfected with 60nM (final concentration) Non-targeting siRNA (Dharmacon, cat # D-001206-13-20, siGENOME Non-Targeting siRNA Pool #1) or BCL2 siRNA (Dharmacon, cat# M-003307-06-0010, SMARTpool: SiGENOME BCL2 siRNA) using TransIT-TKO Transfection Reagent (Mirus, cat# MIR 2154) following the manufacturer’s instructions. Protein was collected and viability was assessed 96 hours post transfection. Data shown are an average and standard deviation of three biological replicates.

Corella A.N., Ordonio M.V., Coleman I., Lucas J.M., Kaipainen A., Nguyen H.M., Sondheim D., Brown L.G., True L., Lee J., MacPherson D., Nghiem P., Gulati R., Morrissey C., Corey E, & Nelson P.S. (2019). Identification of Therapeutic Vulnerabilities in Small Cell Neuroendocrine Prostate Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(7), 1667-1677.

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Evaluating HCMV Infection in Cell Lines

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ARPE-19, MRC5, and HFF-1 cells (2 × 10⁵/well in a 6-well plate) were transfected with SMARTpool: siGENOME CD46 siRNA or siGENOME Non-Targeting siRNA pool #1 (20 nM) (Dharmacon) by RNAiMAX (ThermoFIsher) according to manufacturer’s instructions. 48 h post transfection, cells were infected with TB40/E^{FLAG YFP} or AD169^BADrUL131 (MOI:0.5) and subjected to flow cytometry or western blotting analysis at 48 hpi.

Stein K.R., Gardner T.J., Hernandez R.E., Kraus T.A., Duty J.A., Ubarretxena-Belandia I., Moran T.M, & Tortorella D. (2019). CD46 facilitates entry and dissemination of human cytomegalovirus. Nature Communications, 10, 2699.

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Optimizing NPM1 and H1.5 Knockdown

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Cells growing in 6-well plastic plates were transfected with oligofectamine according to the manufacturer´s instructions (Life Technologies). siRNAs were purchased from Dharmacon including: siGENOME On-TARGETplus SMARTpool human NPM1 (#4869, denoted as siNPM1); siGENOME Human HIST1H1B/H1.5 (#3009) siRNA SMARTpool (denoted as siH1.5#1); and siGENOME non-targeting siRNA pool #1 (D-001206-13-05) denoted as siCtrl. HIST1H1B/H1.5 Stealth siRNA HSS142366 (siH1.5#2) was from ThermoFisherScientific. To reduce NPM1 protein level it was necessary to conduct two rounds of siRNA transfections four days apart.

Holmberg Olausson K., Elsir T., Moazemi Goudarzi K., Nistér M, & Lindström M.S. (2015). NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape. Scientific Reports, 5, 16495.

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Evaluating Transcription Factor Knockdown in ASM Cells

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Mouse ASM cells were plated at a density of 1.1 × 10⁵ cells per well in six-well plates with DMEM containing 10% FBS without any antibiotics. The next day, the cells were transfected with 25 nM siRNAs (siGENOME SMARTpool-Yy1, SMARTpool-Mef2d, SMARTpool-Casp7, SMARTpool-Calp1, or SMARTpool-Calp2, Dharmacon) using the DharmaFECT 1 Transfection Reagent (Dharmacon), according to the manufacturer’s instructions. A siGENOME nontargeting siRNA pool 1 (Dharmacon) was used as a negative control. At 24 hours after transfection, the cells were cultured in the CO₂ medium. At 72 hours after transfection, cells were harvested, and total RNA or protein samples were prepared as described above.

Shigemura M., Lecuona E., Angulo M., Homma T., Rodríguez D.A., Gonzalez-Gonzalez F.J., Welch L.C., Amarelle L., Kim S.J., Kaminski N., Budinger G.R., Solway J, & Sznajder J.I. (2018). Hypercapnia increases airway smooth muscle contractility via caspase-7-mediated miR-133a-RhoA signaling. Science translational medicine, 10(457), eaat1662.

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FYN Knockdown Assay Protocol

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SiGENOME SMARTpool siRNA targeting FYN and siGENOME Non-Targeting siRNA Pool #1 (Dharmacon) were used at 50 nM. Transfection was performed using Dharmafect 1 reagent. Transfection efficiency was confirmed to be ≥90% using siGENOME positive control targeting GAPDH (Supporting Information Fig. S5c). RNA extraction and process outgrowth assay were performed 96h after transfection.

Nelson M., Millican-Slater R., Forrest L.C, & Brackenbury W.J. (2014). The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis. International Journal of Cancer. Journal International du Cancer, 135(10), 2338-2351.

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Ret siRNA Transfection in Neurons

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Neurons (2 DIV) were transfected with siRNA using a scrambled control (siGENOME Non-Targeting siRNA Pool #1; Dharmacon) or Ret (ON-TARGETplus SMARTpool; Dharmacon) siRNA at a concentration of 100 nM via i-Fect (Neuromics) according to the manufacturer’s instructions. Transfection efficiency was determined in all experiments by the cotransfection of a fluorescently labeled non-targeting control siRNA (siGLO RISC-free siRNA; Dharmacon). 48 h after transfection, at which time expression of siGLO was maximal, neurons were treated as described in the figure legends. For immunocytochemistry experiments, neurons were fixed with 4% paraformaldehyde for 5 min, washed, and stained with primary and secondary antibodies and imaged as described in the Fixation, sectioning, and immunostaining of SCG section, with the addition of α-p-c-Jun (9261; 1:500; Cell Signaling Technology). Samples were imaged using 40× magnification with a digital zoom of 1.0 using an Axiovert 200M microscope, with tile scans of 5 × 5 fields to randomly sample non-overlapping areas. AxioVision software was used to stitch images together using 15% overlap between adjacent images. Images were again exported as high-resolution tagged image files, and all figures were created using Adobe Creative Suites.

Donnelly C.R., Gabreski N.A., Suh E.B., Chowdhury M, & Pierchala B.A. (2018). Non-canonical Ret signaling augments p75-mediated cell death in developing sympathetic neurons. The Journal of Cell Biology, 217(9), 3237-3253.

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Investigating FYN gene function via siRNA

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SiGENOME SMARTpool siRNA targeting FYN and siGE-NOME Non-Targeting siRNA Pool #1 (Dharmacon) were used at 50 nM. Transfection was performed using Dharmafect 1 reagent. Transfection efficiency was confirmed to be ≥90% using siGENOME positive control targeting GAPDH (Supporting Information Fig. S5c). RNA extraction and process outgrowth assay were performed 96h after transfection.

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Visualizing EGFR Trafficking in HeLa Cells

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Dharmacon siGENOME non-Targeting siRNA Pool #1 (D-00126-13-05) (100 nM) or siGENOME SMARTpoll human STRIP1 (M-02516091-005) (100 nM) was transfected into HeLa cells (1.0 × 10⁵) with Lipofectamin 2000 (Invitrogen) according to manufacturer’s protocol, cultured for 3 days, and plated on polyethylene-imine-coated cover slips for over night. Cells were pulsed with Alexa Fluor 488 EGF complex (Invitrogen) for 3 min and immediately fixed or rinsed and incubated for 27 min at 37 °C and fixed.

Sakuma C., Kawauchi T., Haraguchi S., Shikanai M., Yamaguchi Y., Gelfand V.I., Luo L., Miura M, & Chihara T. (2014). Drosophila Strip serves as a platform for early endosome organization during axon elongation. Nature communications, 5, 5180.

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