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Sigenome smartpool sirna

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The SiGENOME SMARTpool siRNA is a collection of four individual siRNA duplexes designed to target a specific gene. It is intended for use in RNA interference (RNAi) experiments to knockdown gene expression.

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Lab products found in correlation

Lipofectamine rnaimax, by Thermo Fisher Scientific (5 mentions) Doxycycline, by Merck Group (2 mentions) Transit lt1, by Mirus Bio (2 mentions) Facscelesta, by BD (2 mentions) On targetplus non targeting pool, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Scrambled control sirna, by Thermo Fisher Scientific (1 mentions) Metafectene pro, by Biontex (1 mentions) Sigenome, by Thermo Fisher Scientific (1 mentions) On targetplus, by Thermo Fisher Scientific (1 mentions) Flexitube genesolution, by Qiagen (1 mentions) Lipofectamin rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions) Dharmafect 4 transfection reagent, by Thermo Fisher Scientific (1 mentions) Asp175, by Cell Signaling Technology (1 mentions)

12 protocols using sigenome smartpool sirna

1

ATP6AP2 knockdown and bafilomycin effects

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105 cells/2 ml medium were preincubated in six‐well plates for 2 days to reach 80% cell density before down‐regulation of ATP6AP2 or incubation with bafilomycin 1A (Enzo Life Science, Lörrach, Germany). For microscopy, 104 cells/0.7 ml medium were seeded in four‐chamber cover slides and preincubated for 2 days.
For ATP6AP2 knock‐down, transfection was performed for 6 hrs with a siGENOME SMART pool siRNA against ATP6AP2 mRNA or scrambled control siRNA (Thermo Fisher Scientific Inc, Schwerte, Germany) in a final concentration of 40 nmol/l using Metafectene Pro (Biontex, Planegg/Martinsried, Germany) as transfection reagent. Time‐dependent down‐regulation was validated by qRT‐PCR and Western blot analyses.
Bafilomycin 1A was added to the cells for 1 day in a final concentration of 1 μmol/l. For these experiments, an additional control with 1% DMSO was used.
Wanka H., Lutze P., Staar D., Peters B., Morch A., Vogel L., Chilukoti R.K., Homuth G., Sczodrok J., Bäumgen I, & Peters J. (2017). (Pro)renin receptor (ATP6AP2) depletion arrests As4.1 cells in the G0/G1 phase thereby increasing formation of primary cilia. Journal of Cellular and Molecular Medicine, 21(7), 1394-1410.
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2

RNAi Experiments for Targeted Silencing

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Primary RNAi experiments were performed using siGENOME SMARTpool siRNA (Thermo Scientific). Secondary validation of silencing phenotypes was conducted with individual siRNA oligonucleotides of siGENOME or ON-TARGETplus design (Thermo Scientific), or pooled siRNA from FlexiTube GeneSolution (QIAGEN). Details of individual siRNA sequences as well as non-targeting/negative control siRNA used for normalization and statistical comparisons are provided as supplementary information (tables S6 and S7). Transfections were carried out with Lipofectamine RNAiMAX according to manufacturer’s instructions. Cells were transfected with siRNA at a final concentration of 10 nM (siGENOME), 15 nM (QIAGEN) or 25 nM (ON-TARGETplus) 72 hours prior to assaying the impact of silencing.
Locard-Paulet M., Lim L., Veluscek G., McMahon K., Sinclair J., van Weverwijk A., Worboys J.D., Yuan Y., Isacke C.M, & Jørgensen C. (2016). Phosphoproteomic analysis of tumor-endothelial signaling identifies EPHA2 as a negative regulator of transendothelial migration. Science signaling, 9(414), ra15.
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3

Knockdown of Calcium Channel Genes

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A2780-SP cells (5 × 105 cell/mL) were layered on ultra-low attachment 6-well plates. After seeding the cells, they were transfected with siGENOME SMARTpool siRNA against CACNA1D, CACNA1F, CACNA1H, and non-specific control siRNA (each 100 nM, Thermo Fisher Scientific, USA) using Lipofectamin RNAiMax transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 72 h of transfection, RNA was extracted and reverse transcribed into cDNA. The cDNA was confirmed via knockdown of GAPDH-normalized CACNA1D, CACNA1F, and CACNA1H at each gene level using quantitative real-time PCR.
Lee H., Kim J.W., Kim D.K., Choi D.K., Lee S., Yu J.H., Kwon O.B., Lee J., Lee D.S., Kim J.H, & Min S.H. (2020). Calcium Channels as Novel Therapeutic Targets for Ovarian Cancer Stem Cells. International Journal of Molecular Sciences, 21(7), 2327.
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4

ROCK1 and ROCK2 Knockdown Assay

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SCC-61 and MDA-MB-231 cells were cultured as described previously as well as KD of ROCK1 and ROCK2 with siGENOME SMARTpool siRNA (ThermoScientific) to maximize inhibition while minimizing off-target effects as well as any possible compensatory effects.34-36 KDs were confirmed with Western blotting as described previously.35 A double KD experiment was previously performed to confirm that compensation was not occurring between the ROCK isoforms.35
Jerrell R.J., Leih M.J, & Parekh A. (2017). The ROCK isoforms differentially regulate the morphological characteristics of carcinoma cells. Small GTPases, 11(2), 131-137.
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5

RNA Interference of YY1 in Cells

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Knockdown was performed according to the manufacturer’s instructions. Briefly, 100,000 cells were seeded into each well of a 24 well tissue culture plate. After 24 hrs the cells were transfected with 2 μL of Dharmafect 4 transfection reagent (Thermo Scientific) and 0.05 μM of siGenome SMARTpool siRNA (M-011796-02 for YY1) and harvested after a further 72 hrs.
Chapman A.G., Cotton A.M., Kelsey A.D, & Brown C.J. (2014). Differentially methylated CpG island within human XIST mediates alternative P2 transcription and YY1 binding. BMC Genetics, 15, 89.
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6

YBX1 Knockdown Regulates Gene Expression

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HEK293T cells were first transfected with control or YBX1 siRNA (Dharmacon, siGENOME SMARTpool siRNA) using Lipofectamine RNAiMAX (Thermo Fisher Scientific). 24 hr later, eYFP and mCherry reporter, rtTA expression vector, and pri-miRNA expression plasmids were subsequently cotransfected using TransIT-LT1 (Mirus). 4 hr after the second transfection, HEK293T cells were treated with 1 μg/ml doxycycline (Sigma). 48 hr after the second transfection, flow cytometry analysis was carried out with BD FACS Celesta (BD Biosciences). Data collection was performed using FACS Diva Version 8.0.1. FlowJo version 10.4.1. and R was used for data analysis. After selection of single cell populations by sequential gating by SSC and FSC (Extended Data Fig. 10d), we analyzed about 20,000 eYFP-positive cells (P3 population in Extended Data Fig. 10d). Each experiment included non-transfection controls. The eYFP and mCherry signals of each cell were background normalized by subtracting the mean value plus two standard deviation of signal in non-transfected samples, and binned by eYFP signal levels, as previously described40 (link).
Grigelioniene G., Suzuki H.I., Taylan F., Mirzamohammadi F., Borochowitz Z.U., Ayturk U.M., Tzur S., Horemuzova E., Lindstrand A., Weis M.A., Grigelionis G., Hammarsjö A., Marsk E., Nordgren A., Nordenskjöld M., Eyre D.R., Warman M.L., Nishimura G., Sharp P.A, & Kobayashi T. (2019). Gain-of-function mutation of microRNA-140 in human skeletal dysplasia. Nature medicine, 25(4), 583-590.
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7

YBX1 Knockdown Regulates Gene Expression

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HEK293T cells were first transfected with control or YBX1 siRNA (Dharmacon, siGENOME SMARTpool siRNA) using Lipofectamine RNAiMAX (Thermo Fisher Scientific). 24 hr later, eYFP and mCherry reporter, rtTA expression vector, and pri-miRNA expression plasmids were subsequently cotransfected using TransIT-LT1 (Mirus). 4 hr after the second transfection, HEK293T cells were treated with 1 μg/ml doxycycline (Sigma). 48 hr after the second transfection, flow cytometry analysis was carried out with BD FACS Celesta (BD Biosciences). Data collection was performed using FACS Diva Version 8.0.1. FlowJo version 10.4.1. and R was used for data analysis. After selection of single cell populations by sequential gating by SSC and FSC (Extended Data Fig. 10d), we analyzed about 20,000 eYFP-positive cells (P3 population in Extended Data Fig. 10d). Each experiment included non-transfection controls. The eYFP and mCherry signals of each cell were background normalized by subtracting the mean value plus two standard deviation of signal in non-transfected samples, and binned by eYFP signal levels, as previously described40 (link).
Grigelioniene G., Suzuki H.I., Taylan F., Mirzamohammadi F., Borochowitz Z.U., Ayturk U.M., Tzur S., Horemuzova E., Lindstrand A., Weis M.A., Grigelionis G., Hammarsjö A., Marsk E., Nordgren A., Nordenskjöld M., Eyre D.R., Warman M.L., Nishimura G., Sharp P.A, & Kobayashi T. (2019). Gain-of-function mutation of microRNA-140 in human skeletal dysplasia. Nature medicine, 25(4), 583-590.
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8

Efficient Gene Silencing using siRNA

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Gene silencing was achieved using Dharmacon siGENOME smart pool siRNA using non-targeting siRNA as control (Thermo Fisher Scientific, Lafayette, CO) at a concentration of 100 nM for HCT116 and 25nM for BEAS-2B cells. SiRNA transfections were performed using Dharmafect 4 transfection reagent according to manufacturer instructions. A minimum 90% decrease in all target genes expression was achieved as verified by real-time PCR (Fig. 1A-C). Transfection of siRNA alone had no effect on basal IFN-β gene expression (Fig. 1D).
Lakhdari O., McAllister C.S., Wang M., Minev I., Prince L.S., Eckmann L, & Kagnoff M.F. (2016). TLR3 signaling is downregulated by a MAVS isoform in epithelial cells. Cellular immunology, 310, 205-210.
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9

Beclin-1 Knockdown and Lipoic Acid Treatment

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Small interfering RNA (siRNA) knockdown of beclin-1 was performed using siGENOME SMARTpool siRNA purchased from Thermo Scientific (Waltham, MA). At 40% confluency cells were transfected with 20 nM siRNA using Lipofectamine® RNAimax (Invitrogen, Darmstadt, Germany). Non-sense siRNA was used as negative control. About, 24 h following transfection cells were treated with 1000 µM LA and harvested after 48 h. Beclin-1 knockdown was verified by western blot analysis.
Göder A., Nagel G., Kraus A., Dörsam B., Seiwert N., Kaina B, & Fahrer J. (2015). Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction. Carcinogenesis, 36(8).
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10

STAG2 Knockdown in Cell Lines

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Cells were transfected with 50 nmol/L of STAG2 siRNA (SMARTpool siGENOME siRNA, Thermo Fisher Scientific) or control siRNA (On TargetPlus nonTargeting pool, Thermo Fisher Scientific) using Lipofectamine 2000 (Invitrogen). Forty-eight hours after, a second transfection was performed with same conditions as the first transfection. The cells were cultured for another 72 hours before harvesting for Western blot analysis.
Athans S.R., Krishnan N., Ramakrishnan S., Cortes Gomez E., Lage-Vickers S., Rak M., Kazmierczak Z.I., Ohm J.E., Attwood K., Wang J, & Woloszynska A. (2022). STAG2 Expression is Associated with Adverse Survival Outcomes and Regulates Cell Phenotype in Muscle-invasive Bladder Cancer. Cancer Research Communications, 2(10), 1129-1143.
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