MDA-MB-231 cells (300,000 cells/well) were seeded in 6-well plate. After 24 h, the cells were transfected for 48 h with 20 nM siRNA (KLK7: S11267) or 50 nM siRNA (CCR10: S194449; OXTR: S9946; SSTR3: S13503; TACR2: S13711; Neg Ctrl: AM17110; Life Technologies) using silentFect reagent according to the manufacturer’s instructions (BIO-RAD). RT-qPCR was performed to determine the extent of gene knockdown.
Silentfect reagent
Manufactured by Bio-Rad
Sourced in United States, Canada
SiLentFect is a cationic lipid-based transfection reagent that facilitates the delivery of small interfering RNA (siRNA) or other nucleic acids into a variety of cell types. It enables efficient gene silencing without significant cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Allstar control sirna, by Qiagen (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Luciferin reagent, by Promega (1 mentions)
Lipofectmine 2000, by Thermo Fisher Scientific (1 mentions)
Smartpool, by Thermo Fisher Scientific (1 mentions)
Wst 1 cell viability assay kit, by Daeil Lab Service (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
On targetplus smartpool sirna, by Horizon Discovery (1 mentions)
Opti mem media, by Thermo Fisher Scientific (1 mentions)
14 protocols using silentfect reagent
1
siRNA Knockdown of Cell Signaling Proteins
2
Knockdown of TDP-43 in Neuroblastoma and HeLa Cells
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M17 neuroblastoma and HeLa cells were cultured in OptiMEM (Invitrogen), and when confluent, 1.75 × 104 cells/well plated to 24-well plates on coverslips. TDP-43 was knocked down for 72 h with 20 nM TARDBP siRNA (QIAGEN) (Prudencio et al, 2012 (link)) or All Star control siRNA (QIAGEN) and siLentFect reagent (Bio-Rad) and fixed in 4% PFA in DEPC-treated PBS and permeabilized. Nuclease treatment of HeLa cells was performed at 37°C for 30 min with 100 U/ml of RNase-free DNase I and RNase A (QIAGEN), ShortCut RNase III (NEB) or ultrapure benzonase nuclease (Sigma) each in their recommended buffers. Protein Block Serum Free (Dako) was used for blocking. Primary antibody incubation was done overnight in Antibody Diluent (Dako) with 0.25 μl/ml RNase OUT and 1:1,000 anti-TDP-43-Cterm (ProteinTech) and 1:1,000 J2, and secondary antibodies were 1:1,000 (donkey) anti-rabbit-AF488 and anti-mouse-AF568 (Alexafluor). Coverslips were counterstained with 0.1 μg/ml Hoechst. Images were taken by Zeiss AxioImager Z1 with Apotome at 63× with fixed exposures times.
3
Gene Expression Regulation by Transfection
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Plasmid DNA were transfected into cells using Lipofectmine 2000 (Invitrogen), while siRNA was transfected by siLentFect reagent (Bio-Rad). Luciferase activities were determined using the luciferin reagent (Promega) according to the manufacturer's protocol. Transfection efficiency was normalized to Renilla luciferase activities.
4
Silencing of Estrogen Signaling Pathway Genes
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Cells were maintained in hormone-depleted media for three days before transfection. Cells were seeded and transfected when attached. A SMARTpool of four siRNAs was used against CA12 and TFAP2C and two different ON-TARGETplus siRNAs against ESR1, FOXA1 or GATA3 (Dharmacon, Lafayette, CO, USA). One ON-TARGETplus Non-Targeting siRNA (“si-Control”, Dharmacon, Lafayette, CO, USA) was used as a negative control. Transfection was performed with each siRNA (40 nM) using the SilentFect reagent (BioRad, Hercules, CA, USA) for a total duration of 72 h. Cells were then treated or not with E2 (25 nM) or with vehicle (EtOH 0.024%), 24 h before cell collection for subsequent RNA and protein extractions. Sequences of all siRNAs are provided in Supplementary Table S3 .
5
Investigating Cell Invasion Pathways
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TIME cells were transiently transfected with 5 nM of siRNAs for scrambled control, HYAL2 or CD44 for 24 h, followed by subculture on plastic dish or on Matrigel for another 16 h. In some experiments, prior to seeding them on Matrigel, cells were pre-treated for 1 h with 36 µM of the cell-permeable NF-kB SN50 inhibitor peptide or the control inactive peptide SN50M (both from Calbiochem). All siRNAs were purchased from Dharmacon (ONTarget SMARTpool Plus) and transfected into the cells using SilentFect reagent (Biorad) according to the manufacturer's instructions. Knockdown efficiency was routinely checked at the mRNA and/or protein levels.
6
Investigating LYN's Role in Cell Proliferation and Motility
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siRNAs against LYN and a non-targeting SMART pool were purchased from Thermo Scientific. FLAG-LYN and FLAG overexpression tags were constructed using standard molecular cloning methods. Briefly, 2 × 106 cells were transfected with siRNAs (20 nmol) or overexpression constructs (10 μg) for 72 h using siLentFect reagent (Bio-Rad) or Lipofectamine (Invitrogen), respectively. The effect of gene silencing or overexpression on cell proliferation was measured using the WST-1 cell viability assay kit (Daeil Lab Service). Motility and invasion were quantified by QCMTM 24-well cell migration or invasion assays according to the manufacturer's instructions (Millipore). All assays were performed in triplicate, and the means and standard errors are shown.
7
CTSD siRNA Knockdown in MDA-MB-231 Cells
8
Knockdown of Regulatory Proteins for Nuclear Fractionation
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For the knockdown of clathrin, cells were seeded in 100-mm Petri dishes and transfected the next day at ∼50% confluence with 20 nM clathrin heavy chain Silencer Select siRNA (Invitrogen) using 5 µl of SilentFect reagent (BioRad) in 10% FBS in DMEM. The next day, the media were replaced with 0.1% FBS in DMEM, and on day 2, cells were replated and retransfected with 20 nM siRNA, serum starved for 3 d, stimulated with PDGF-BB, and collected for nuclear fractionation. As control knockdown, stealth RNAi negative control #12935112 (Invitrogen) was used. For the knockdown of β-importin, cells were seeded as above and transfected with a mix of three siRNAs (4 nM each) from the Tri-silencer siRNA kit (Origene) using 5 µl of SilentFect reagent; the negative control was used as provided in the kit. The medium was replaced with 0.1% FBS in DMEM, and cells were stimulated with PDGF at 6 d after knockdown and collected for nuclear fractionation. For the knockdown of Arf-1, Fer, TMF-1, and Brg-1, cells were transfected with Tri-silencer siRNA kits in the same ways as for Kpn, but analyzed at 3 d after knockdown. Levels of the knockdown were analyzed by immunoblotting (clathrin, Fer, TMF-1, Brg-1, and β-importin) or quantitative PCR (Arf-1 and TMF-1).
9
MRTF-A Knockdown in CF Cells
10
Silencing NGF, p75NTR and TRKA Signaling
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Approximately 1.5×105 Mia PaCa2 or BxPC-3 cells were plated in each well of a 6-well plate in serum free media (SFM) and allowed to attach overnight. On the next day, siLentFect reagent (BioRad, Hercules, CA) was used to deliver the SMARTPool siRNA (Dharmacon RNAi Technologies, Thermo Scientific, Pittsburg, PA) for NGF, p75NTR and TRKA at a 100nM concentration. A non-targeting siRNA as a negative control and a siRNA against the UBB protein as a positive control for transfection were also transfected into the cells using the manufacturer’s recommended protocol. Briefly, 4μl of siLentFect and 100nM siRNA were mixed together in equal volumes, in serum free medium (SFM) in poly-styrene tubes and allowed to incubate at room temperature for 30 minutes. All the media from the wells was aspirated off and 500 μl of the siRNA—siLentFect mix was added to each well along with 1.5ml of media. After incubation for 72 hours cells were assayed for NGF, p75NTR and TRKA protein expression and used in the Boyden Chamber migration assay and Dorsal root ganglion co-culture assay.
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