COV362 cells were transfected with 5 nM non-targeting pool siRNA (catalog no. D-001810-10-05), NOX4 SMARTpool siRNA (catalog no. L-010194-00), or NOX4 #07 individual siRNA (catalog no. J-010194-07) from Dharmacon, utilizing RNAiMAX Lipofectamine™ transfection reagent (catalog no. 13778; Invitrogen). Transfected cells were harvested 96 h post-treatment, and silencing efficiency was evaluated at both the RNA (qPCR) and protein level (immunoblot).
Non targeting sirna pool
Manufactured by Horizon Discovery
Sourced in United States, United Kingdom
The Non-targeting siRNA pool is a collection of small interfering RNA (siRNA) molecules that do not target any known gene. This pool serves as a control for experiments involving RNA interference (RNAi) to help assess the specificity of targeted gene silencing.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (6 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (5 mentions)
Oligofectamine, by Thermo Fisher Scientific (4 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (3 mentions)
Sirna buffer, by Horizon Discovery (2 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Small interfering rna duplexes targeting, by Horizon Discovery (2 mentions)
On targetplus sirna, by Horizon Discovery (2 mentions)
Mouse fbxw7 specific smart pool sirna, by Horizon Discovery (2 mentions)
Mouse talpid3 specific smart pool sirna, by Horizon Discovery (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Universal negative sirna, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
33 protocols using non targeting sirna pool
1
Silencing NOX4 in COV362 Cells
2
Knockdown of LEF1 in MCF7-DR Cells
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MCF7-DR cells were plated in 6-well plates at a density of 1 × 105 cells per well. The following day, SmartPool LEF1 siRNA (#51176) or nontargeting pool siRNA (Dharmacon, Lafayette, CO, USA) was added to cells with Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Twelve hours after the transfection, the medium was changed, and the cells were incubated for 60 more hours. Total RNA was extracted from cell lines using QIAzol reagent and the miRNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. Reverse transcription from 1 μg of total RNA was carried out using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Real-time PCR analysis was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA) on a StepOne Real-Time PCR System (Applied Biosciences, Waltham, MA, USA). The TaqMan probes used in this study were LEF1 (Hs01547250_m1), CAV1 (Hs00971716_m1), ABCG2 (Hs01053790_m1), VIM (Hs00958111_m1), ACTA2 (Hs00426835_g1), TGFB2 (Hs00234244_m1) and ACTB (Hs03023880_g1). The expression levels were normalized to those of ACTB, and the relative fold changes in mRNA expression levels were calculated using the formula 2−ΔΔCt.
3
Investigating AMPK α2 and PEA15 in Breast Cancer
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Primary HMECs were transfected with Accell small interfering RNA (siRNA) oligos (GE Healthcare Dharmacon, Little Chalfont, UK) targeting AMPK α2 and PEA15 as per the manufacturer’s protocol; nontargeting pool siRNA (GE Healthcare Dharmacon) was used as control siRNA. Breast cancer cell lines were transfected with specific siRNA oligos targeting AMPK α2 and PEA15 (Cell Signaling Technology); universal negative siRNA (Sigma-Aldrich) was used as control. Adherent Michigan Cancer Foundation 7 breast cancer cell line (MCF7) and breast cancer cell line derived from metastatic site (pleural effusion) (MDAMB231) were transfected with 20 nM siRNA using oligofectamine (Invitrogen). After 48 hrs of siRNA transfection, the cells were trypsinized and seeded in methylcellulose for further experimentation. AMPK α2 knockdown stable cells were generated by transfecting MDAMB231 and BT 474 cells with a pool of four short hairpin RNA (shRNA) constructs targeting AMPK α2 (pRFP-C-RS-PRKAA2) or scrambled (HuSH-29 shRNA vectors; (Origene Technologies, Rockville, MD, USA) using Lipofectamine-2000 (Invitrogen). Stable cells were generated using puromycin (0.5 μg/ml) selection followed by flow cytometer-based sorting (MoFlo; Beckman Coulter, Brea, CA, USA) for RFP expression (encoded by the vector) and were expanded and frozen for future use. Knockdown was confirmed by immunoblotting.
4
Silencing VEGFR2 in Cultured Neurons
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As in our previous study [34 (link)], ON-TARGET plus SMART pool for mouse VEGFR2 siRNA (#L-040634-00-0020) and Non-Targeting Pool siRNA (#D-001810-10-05) were obtained from Dharmacon (Cambridge, UK). The siRNA was resuspended in 1× siRNA buffer, which was diluted from 5× siRNA buffer (Dharmacon) with RNase-free water, to obtain a 20 μM stock concentration. Cultured neurons were transfected with Lipofectamine RNAiMAX reagent (#13778-075, Invitrogen) according to the manufacturer’s transfection protocol; a final volume 0.3 μL/well in 96-well plate or 7.5 μL/well in 6-well plate was used. Briefly, the cultured neurons were incubated with the siRNA-duplex-Lipofectamine RNAiMAX complex at a final siRNA concentration of 10 nM for 48 h at 37 °C in a CO2 incubator.
5
Evaluation of IL-13Rα2 Knockdown on Cell Migration
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Gene knockdown by siRNA was accomplished by transfecting Stealth RNAi duplexes (Invitrogen Life Technologies) pre-designed for human IL-13Rα2 or ON-TARGETplus SMARTPool human IL-13Rα2 siRNA and non-targeting pool siRNA (Dharmacon, CO, USA) at a final concentration of 3-10 nM using Lipofectamine RNAiMax (Invitrogen Life Technologies) according to manufacturer’s instruction. Proliferation assay using CCK-8 was performed at the indicated time points. For migration or invasion assays, the transfected cells were seeded to the top well of the 8 μm migration (BD Biosciences) or Matrigel invasion chamber (BD Biosciences). Migrated cells were fixed with 4% paraformaldehyde (PFA) after 8 h and mounted in mounting medium containing propidium iodide (PI; 100 µg/ml) and RNase (2 mg/ml; Sigma-Aldrich). YKL-40 i.e., CHI3L1 was from R&D Systems and IL-13 was from Peprotech. The percentage of migrated cells was subsequently quantified by counting the number of PI-stained nuclei cells in the bottom side of the membrane from at least five random fields at ×200 magnification.
6
Cx43 siRNA Knockdown in bEnd.3 and ANS4-GFP Cells
7
Silencing FRG1 Expression in Cell Line
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The cell line that stably expresses WT FRG1 was treated with 200 nM non-targeting pool siRNA (Dharmacon; D-001810-10-05) and 150 or 200 nM U2AF65 siRNA (Dharmacon)59 (link) using RNAiMAX (Invitrogen) according to manufacturer’s instructions. The siRNA sequence is 5′-GCACGGUGGACUGAUUCGUdTdT-3′. Experiments were performed 48 h after transfection.
8
MMP9 Knockdown in Co-Culture
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OVCA433 were seeded at 1,300 cells/cm2 and allowed to attach overnight (cell density was adjusted to account for increased culture time in device during siRNA treatment). Coverslips were removed, cells were washed with PBS, and cells were treated for 24 hours with 25 nM ON-TARGETplus MMP9 or non-targeting pool siRNA (Dharmacon; Lafayette, CO). THP-1 were differentiated as previously stated for 72 hours, washed with PBS, and treated for 24 hours with 50 nM ON-TARGETplus MMP9 or non-targeting pool siRNA. Each cell type was allowed to recover in SFM for 24 hours prior to use in co-cultures.
9
Gastric Cancer Cell Line Knockdown
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Cell culture. The poorly differentiated gastric adenocarcinoma cell line NUGC3 (American Type Culture Collection, Manassas, VA, USA) was routinely cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (both form HyClone; GE Healthcare Life Sciences, Logan, UT, USA). When the cells were 80-90% confluent, subculturing of the cells were performed.
Short interfering RNA (siRNA/si) transfection. NUGC3 cells were plated in 6-well plates at a density of 2x10 5 cells/well. YBX1, ABCC2 and ABBC3 ON-TARGETplus SMARTpool siRNAs, as well as non-targeting pool siRNA were obtained from GE Healthcare Dharmacon, Inc. (Lafayette, CO, USA), while siRNA for STAT3 was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Table I list the sequences of siRNAs used in the present study. NUGC3 cells were then transfected with 20 nM of siNegative, siYB-1, siSTAT3, siABCC2 or siABCC3 using DharmaFECT1 Transfection reagent (GE Healthcare Dharmacon, Inc.). Following an overnight incubation with siRNAs, culture medium was replaced prior to further incubation. The transfected cells were then collected at 48 h post transfection for RNA isolation and 72 h post transfection for protein isolation. For double knockdown of STAT3 and YB-1, each siRNA was used at a final concentration of 20 nM.
Short interfering RNA (siRNA/si) transfection. NUGC3 cells were plated in 6-well plates at a density of 2x10 5 cells/well. YBX1, ABCC2 and ABBC3 ON-TARGETplus SMARTpool siRNAs, as well as non-targeting pool siRNA were obtained from GE Healthcare Dharmacon, Inc. (Lafayette, CO, USA), while siRNA for STAT3 was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Table I list the sequences of siRNAs used in the present study. NUGC3 cells were then transfected with 20 nM of siNegative, siYB-1, siSTAT3, siABCC2 or siABCC3 using DharmaFECT1 Transfection reagent (GE Healthcare Dharmacon, Inc.). Following an overnight incubation with siRNAs, culture medium was replaced prior to further incubation. The transfected cells were then collected at 48 h post transfection for RNA isolation and 72 h post transfection for protein isolation. For double knockdown of STAT3 and YB-1, each siRNA was used at a final concentration of 20 nM.
10
p5RHH Peptide-Mediated siRNA Delivery
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p5RHH peptide (VLTTGLPALISWIRRRHRRHC) provided by Genscript was dissolved at 20 mM in DNase-, RNase-, and protease- free sterile purified water (Cellgro) and stored in 10 μL aliquots at −80 °C before use. Human RELA SMART pool SiRNAs (a mixture of 4 different siRNAs at a ratio of 1:1:1:1; Cat# M-003533-02, Dharmacon) and ON-TARGETplus control Non-targeting pool siRNAs (Cat# D-001810-05, Dharmacon) were dissolved at 20 uM in DNase, RNase-, and protease- free sterile purified water (Cellgro) and stored in 10 μL aliquots at −80 °C until use.
For cell culture p5RHH siRNA NP was prepared by diluting p5RHH peptide (20 mM) 1:400 in Opti-MEM (Thermo Fisher Scientific), vortexed for 30 s followed by the addition of appropriate volume of RELA SMART pool or ON-TARGETplus control Non-targeting pool stock to achieve a peptide to siRNA ratio of 100 to 1. The mixture was incubated at 37 °C for 40 min with shaking.
For cartilage explant culture, the p5RHH siRNA NP was freshly prepared by mixing p5RHH peptide stock (20 mM) and siRNA (20 uM) to achieve a peptide to siRNA ratio of 1 to 10 in HBSS with Ca2+ and Mg2+ (Gibco, Life Technologies). The mixture was incubated on ice for 10 min and added directly to cartilage explants.
For cell culture p5RHH siRNA NP was prepared by diluting p5RHH peptide (20 mM) 1:400 in Opti-MEM (Thermo Fisher Scientific), vortexed for 30 s followed by the addition of appropriate volume of RELA SMART pool or ON-TARGETplus control Non-targeting pool stock to achieve a peptide to siRNA ratio of 100 to 1. The mixture was incubated at 37 °C for 40 min with shaking.
For cartilage explant culture, the p5RHH siRNA NP was freshly prepared by mixing p5RHH peptide stock (20 mM) and siRNA (20 uM) to achieve a peptide to siRNA ratio of 1 to 10 in HBSS with Ca2+ and Mg2+ (Gibco, Life Technologies). The mixture was incubated on ice for 10 min and added directly to cartilage explants.
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