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Silentfect lipid

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SiLentFect lipid is a cationic lipid-based reagent designed for the transfection of small interfering RNA (siRNA) into mammalian cells. It facilitates the delivery of siRNA into the cells, allowing for the silencing of target gene expression.

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Lab products found in correlation

Scrambled sirna, by Qiagen (1 mentions) Opti mem medium, by Thermo Fisher Scientific (1 mentions) Flavopiridol, by Merck Group (1 mentions) Multiclamp commander, by Molecular Devices (1 mentions) Multiclamp 700b amplifier, by Molecular Devices (1 mentions) Borosilicate glass capillaries, by Harvard Apparatus (1 mentions) Nucleocuvette, by Lonza (1 mentions) Trilencer 27 human sirna, by OriGene (1 mentions) Human amphiregulin duoset elisa, by R&D Systems (1 mentions) Amaxa nucleofector 96 well shuttle system, by Lonza (1 mentions)

6 protocols using silentfect lipid

1

Transfection of PRR siRNA in Cells

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Transfection of PRR siRNA or scrambled siRNA was performed in six-well plate using the siLentFect lipid reagent (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. One hundred µmol of Accell mouse PRR siRNA - SMARTpool (Thermo Scientific Dharmacon Research Inc, USA, target sequences: 5′-CGAAUAGAUUGAAUUUUCC-3′; 5′-CGGUAUACCUUAAGUUUAU-3′; 5′-UGGUUUAGUAGAGAUAUUA-3′; 5′-GGACCAUCCUUGAGGCAAA-3′) was used for each well. After 6 h incubation in transfection reagent, the cells were then switched to normal medium for overnight recovery and ready for experiment. A scrambled siRNA (QIAGEN, Valencia, CA, target sequences: 5′-AATTCTCCGAACGTGTCACGT-3′), which was confirmed as non-silencing double-stranded RNA, was used as control for siRNA experiments
Li C, & Siragy H.M. (2014). High Glucose Induces Podocyte Injury via Enhanced (Pro)renin Receptor-Wnt-β-Catenin-Snail Signaling Pathway. PLoS ONE, 9(2), e89233.
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2

Atg5 Knockdown Using siRNA

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Small interfering RNA (siRNA) against Atg5 (Stealth Atg5-siRNA; MSS247019, MSS247020, and MSS247021) and negative control siRNA (Stealth RNAi Negative Control kit; 12935110) were purchased from Invitrogen (Carlsbad, CA, USA). Cells were seeded in 6-well plates one day before experiments. The growth medium was replaced with 800 μl OPTI-MEM medium (Invitrogen) before siRNA transfection. OPTI-MEM medium (200 μl) contained siLentFect Lipid (BIO-RAD, Philadelphia, PA) and 80 nM Atg5 siRNA or negative siRNA was added to each well. After 6 h of transfection, OPTI-MEM medium was replaced with 1 ml growth medium.
Chiang C.K., Wang C.C., Lu T.F., Huang K.H., Sheu M.L., Liu S.H, & Hung K.Y. (2016). Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury. Scientific Reports, 6, 34167.
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3

Targeting Met Receptor with siRNAs

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Several Met-specific siRNAs—siMet1251 (human specific), siMet4268 (cross species), and two 3′ untranslated region (siMet3′-1,2)—as well as a scrambled siRNA, were ordered from Integrated DNA Technologies (Coralville, IA) and annealed to generate double-stranded siRNAs. An additional Met-targeted siRNA, siMet565, was tested, but it failed to block Met expression and was used as a second control siRNA (shMetMut). siRNA sequences were as follows: siMet1251, 5′-AACCAATG­GA­TCGATCTGCCA-3′; siMet4268, 5′-AATGTGT­CG­CTCCGTATCC­TT-3′; siMet565 (shMetMut), 5′-AAGGACCGGTTCATCAACTTC-3′; siMet3′-1, 5′-CTACCAGGGTTCAAGAGCATGAACGC-3′, siMet3′-2, 5′- CACCCATTAGGTAAACATTCCCUTT-3′; and scrambled, 5′-ACTACCGTTGTTATAGGTG-3′. siRNAs between 5 and 30 nM were introduced into PrECs using SiLentfect lipid from Bio-Rad (Hercules, CA), and the lowest concentration of siRNA giving the most effective knockdown was subsequently used. Control studies using fluorescently labeled control siRNAs indicated >90% transfection efficiency in PrECs.
Tesfay L., Schulz V.V., Frank S.B., Lamb L.E, & Miranti C.K. (2016). Receptor tyrosine kinase Met promotes cell survival via kinase-independent maintenance of integrin α3β1. Molecular Biology of the Cell, 27(15), 2493-2504.
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4

Regulation of H2A.Z by siRNA and Flavopiridol

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MCF7 cells were cultured in DMEM plus 10 % fetal bovine serum. For siRNA knockdown experiments, 10 μM scramble or H2A.Z siRNA (5′-CAGGACUCUAAAUACUCUATT-3′) (Qiagen) were transfected with silentFect™ lipid (Biorad). The cells were collected for western blotting and ChIP 48 h after transfection.
For pause-release inhibition experiments, MCF7 cells were treated with 500 nM flavopiridol (Sigma) or vehicle (DMSO) for 2 h and then collected for H2A.Z and histone H3 ChIP-qPCR.
Day D.S., Zhang B., Stevens S.M., Ferrari F., Larschan E.N., Park P.J, & Pu W.T. (2016). Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types. Genome Biology, 17, 120.
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5

Characterizing CaV2.1 Channel Variants

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For CaV2.1, HEK293 cells were transfected with 0.5 µg CaV2.1-WT or 0.5 µg CaV2.1-V1686M and co-transfected with 0.5 µg CaVβ2, 0.5 µg CaVα2δ1 and 0.2 µg EGFP. All transfections were done using siLentFect™ Lipid (Bio-Rad, Copenhagen, Denmark) according to manufacturer’s instructions. Patch-clamp experiments were performed at room temperature. Currents were measured 72 h after transfection from single fluorescent CHO or HEK cells using a MultiClamp 700B amplifier and MultiClamp Commander (Molecular Devices, Axon Instruments, Sunnyvale, California, United States). The cells were superfused with an extracellular solution containing the following (in mM): 140 TEA-Cl, 3 CsCl, 2.5 CaCl2, 1.2 MgCl2, 10 HEPES and 10 Glucose, pH adjusted to 7.4 with NaOH. Pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, Holliston, United States) using a DMZ Universal Puller (Zeitz Instruments, Martinsried, Germany) and had a resistance of 4.0-6.0 MΩ when filled with intracellular solution containing the following (in mmol/L): 140 CsCl, 1 EGTA, 4 Na2ATP, 0.1 Na3GTP and 10 HEPES, pH adjusted to 7.2 with CsOH. Data were acquired using a Digidata 1,440 Converter and the software pClamp 10.4 Commander (Molecular Devices).
Vad O.B., Yan Y., Denti F., Ahlberg G., Refsgaard L., Bomholtz S.H., Santos J.L., Rasmussen S., Haunsø S., Svendsen J.H., Christophersen I.E., Schmitt N., Olesen M.S, & Bentzen B.H. (2022). Whole-Exome Sequencing Implicates Neuronal Calcium Channel with Familial Atrial Fibrillation. Frontiers in Genetics, 13, 806429.
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6

Silencing AREG Secretion in A549 Cells

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T cells were resuspended in high-efficiency buffer (140 mM sodium phosphate (pH 7.2), 5 mM KCl and 10 mM MgCl2) with siRNA (Trilencer-27 Human siRNA, Origene). Cells were transferred to Nucleocuvettes (Lonza) and electroporated using program EO-115 on the Amaxa Nucleofector 96-well Shuttle System (Lonza). After electroporation, cells were transferred to prewarmed complete medium.
A549 cells (1 × 105 cells per well) were seeded in a 12-well plate containing complete medium and transfected with siRNA using siLentfect Lipid (BioRad) reagent. To quantify AREG secretion, medium was replaced 48 h following lipofection and collected 24 h later, for analysis by the Human Amphiregulin DuoSet ELISA (R&D Systems).
Kubo S., Fritz J.M., Raquer-McKay H.M., Kataria R., Vujkovic-Cvijin I., Al-Shaibi A., Yao Y., Zheng L., Zou J., Waldman A.D., Jing X., Farley T.K., Park A.Y., Oler A.J., Charles A.K., Makhlouf M., AbouMoussa E.H., Hasnah R., Saraiva L.R., Ganesan S., Al-Subaiey A.A., Matthews H., Flano E., Lee H.H., Freeman A.F., Sefer A.P., Sayar E., Çakır E., Karakoc-Aydiner E., Baris S., Belkaid Y., Ozen A., Lo B, & Lenardo M.J. (2021). Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease. Nature immunology, 23(1), 75-85.
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