Scrambled sirna

Manufactured by Merck Group

Sourced in United States

Scrambled siRNA is a laboratory reagent used in molecular biology research. It is a short, double-stranded RNA molecule that does not target any specific gene. Scrambled siRNA is often used as a control in RNA interference (RNAi) experiments to assess the specificity of gene silencing effects.

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38 protocols using scrambled sirna

Transfection and Analysis of HeLa and XTC Cells

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HeLa cells (Leibniz Institute Collections of Microorganisms and Cell Culture, DSMZ, Germany) were cultured in DMEM supplemented with 10% fetal calf serum (Life Technologies, CA, USA) at 37 °C in a humidified atmosphere of 10% CO₂ and transfected as indicated using Rotifect (Carl Roth, Germany) according to the manufacturer’s instructions.
Xenopus embryonic fibroblasts (XTC, a kind gift from Ana Losada) were cultured in 67% DMEM/H2O supplemented with 10% fetal calf serum at 25 °C in a humidified atmosphere of 5% CO₂ and transfected as indicated using Viafect (Promega, USA). Plasmids for transfection were as indicated and 10–20 Hela or XTC cells were analyzed for each condition per experiment.
For siRNA experiments, a combination of three siRNA targeted against BTF3 were transfected as described above. A standard siRNA (scrambled siRNA from Sigma) was used as a control. Catalog number for these commercial siRNA against BTF3 are: SASI_Hs01_00124567; SASI_Hs02_00308337; SASI_Hs01_00124566. Cells were collected 48 h after transfection and processed for qPCR, western blot, or Immunohistochemestry experiments. As described in the respective sections.
All cell lines were tested for mycoplasma contamination.

Kotini M., Barriga E.H., Leslie J., Gentzel M., Rauschenberger V., Schambony A, & Mayor R. (2018). Gap junction protein Connexin-43 is a direct transcriptional regulator of N-cadherin in vivo. Nature Communications, 9, 3846.

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Murine Lung Epithelial Cell Culture

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The murine lung epithelial cell line (MLE12 cells) (American Type Culture Collection, Manassas, VA, USA) was cultured in HITES medium complemented with 10% fetal bovine serum. Cells were maintained in a 37°C incubator in the presence of 5% CO₂. V5 antibody was purchased from Invitrogen (Carlsbad, CA). β-actin antibody, scrambled siRNA, LPA1 siRNA, control shRNA, TrkA shRNA, batimastat (BB94), and LPA were from Sigma Aldrich (St. Louis, MO). GM6001 was from Enzo Life Science (Farmingdale, NY). Antibodies to phospho (pY674/Y675)-TrkA and Myc tag were from Cell Signaling Technology (Danvers, MA). Antibody to TrkA was from EMD MilliPore (Billerica, MA). Antibodies to phospho (p)-Erk1/2, Erk1/2, and TrkA inhibitor (TrkAi) were from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies and ECL kit for detection of proteins by Western blotting was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). All other reagents were of analytical grade.

Nan L., Wei J., Jacko A.M., Culley M.K., Zhao J., Natarajan V., Ma H, & Zhao Y. (2015). Cross-talk between lysophosphatidic acid receptor 1 and tropomyosin receptor kinase A promotes lung epithelial cell migration. Biochimica et biophysica acta, 1863(2), 229-235.

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Silencing LDHA Enzyme Activity

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siRNA oligonucleotides for LDHA were purchased from Sigma-Aldrich, with a scrambled siRNA (Sigma-Aldrich) used as a control. A vector containing wild-type LDHA was purchased from Origene (RC209378; Rockville, MD, USA). Transfection was performed using the Oligofectamine Transfection Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. At 48 h post-transfection, whole-cell lysates were prepared for further analysis.

FENG L., E L.L., SOLOVEIV M.M., WANG D.S., ZHANG B., DONG Y.W, & LIU H.C. (2015). Synergistic cytotoxicity of cisplatin and Taxol in overcoming Taxol resistance through the inhibition of LDHA in oral squamous cell carcinoma. Oncology Letters, 9(4), 1827-1832.

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Lipofectamine 2000 Transfection of ErbB2 and LDHA

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Transfection was performed using the Lipofectamine 2000 Transfection reagent (Invitrogen) according to the manufacturer’s protocol. Overexpression vectors containing wild-type ErbB2 (RC212583) and LDHA (RC228293) were purchased from www.origene.com. The siRNA oligonucleotides for ErbB2 was purchased from Sigma-Aldrich, with a scrambled siRNA (Sigma-Aldrich) used as a control. Forty-eight hours after transfection, cells were collected or whole-cell lysates were prepared for further analysis.

Tang X.Y., Zheng W., Ding M., Guo K.J., Yuan F., Feng H., Deng B., Sun W., Hou Y, & Gao L. (2016). miR-125b acts as a tumor suppressor in chondrosarcoma cells by the sensitization to doxorubicin through direct targeting the ErbB2-regulated glucose metabolism. Drug Design, Development and Therapy, 10, 571-583.

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Endothelial Cell Culture and Analysis

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Human lung microvascular endothelial cells (HLMVECs, Lonza) were cultured at 37°C in an atmosphere of 5% CO₂ with EGM-2 medium (Lonza) containing 25 mL FBS (5%), 0.5 mL hEGF, 2.0 mL hFGF-β, 0.5 mL VEGF, 0.5 mL ascorbic acid, 0.2 mL hydrocortisone, 0.5 mL R3-IGF-1, and 0.5 mL gentamycin. Phospho (T18/S19)-MLC, MLC, antibodies, and cell lysis buffer were obtained from Cell Signaling. Phospho (Y658)-VE-cadherin antibody was purchased from Invitrogen. VE-cadherin antibody was from Santa Crus Biotechnology. β-Actin antibody, scrambled siRNA, LPA1 siRNA, and LPA were from Sigma Aldrich. LPA1 antibody was obtained from Proteintech. AM966 was from Apex Bio. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies, ECL kit, and SDS-PAGE for western blotting were purchased from Bio-Rad Laboratories, Inc. For immunostaining, anti-mouse Alexa-488, anti-rabbit Alexa-568, and DAPI were from Invitrogen. Transfection reagent FuGENE HD was from Promega. All other reagents were of analytical grade.

Cai J., Wei J., Li S., Suber T, & Zhao J. (2017). AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin. Mediators of Inflammation, 2017, 6893560.

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Knockdown of ERK1 and ERK2 in Monocytes

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In knockdown experiments, small interfering RNA (siRNA) against ERK1 and scrambled siRNA were purchased from Sigma-Aldrich while Signalsilencer ® p42 MAPK (ERK2) siRNA II was purchased from Cell Signaling Company. siRNAs were delivered in monocytes by Amaxa nucleofector I (Lonza). 5′ –GACCGGAUGUUAACCUUUA-3′ and 5′-AAGCUGACCCUGAAGUUCA-3′ sequences were used to knock-down ERK1 and as scrambled control (32 (link),33 (link)). For nucleofection, 5×10⁶ monocytes were re-suspended in 100 μl of nucleofection solution containing 150 pmol siRNA for ERK1 and scrambled control, while siRNA against ERK2 was used according the manufacturer protocol. Nucleofection was performed with the Y-01 program. Immediately after nucleofection, monocytes were resuspended in RPMI medium supplemented with 10% FBS and left to recover overnight in polypropylene culture tubes to avoid adherence. The next morning, monocytes were counted with trypan blue showing that 90% of cells were viable. Then cells were treated with 1 μg/ml LPS for 30 minutes followed by 5 mM ATP for another 30 minutes. Released IL-18 in cell culture medium was measured using ELISA while cells were lysed and analyzed for proteins.

Ghonime M.G., Shamaa O.R., Das S., Eldomany R.A., Fernandes-Alnemri T., Alnemri E.S., Gavrilin M.A, & Wewers M.D. (2014). Inflammasome priming by LPS is dependent upon ERK signaling and proteasome function. Journal of immunology (Baltimore, Md. : 1950), 192(8), 3881-3888.

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CCR3 Silencing in Rat Hippocampal Neurons

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Primary rat hippocampal neurons were seeded in 12-well plates at 3.0 × 10⁵ cells/well. siRNA knockdown targeted rat CCR3 using FlexiTube GeneSoultion GS117027 for Ccr3 (1027416, QIAGEN). Hippocampal neurons were transfected with 10 nmol/L by using HiPerFect siRNA Transfection reagent (301704, QIAGEN) following the manufacturer’s instructions. Scrambled siRNA (SIC001, Sigma) was used as the negative control.

Tashiro R., Ozaki D., Bautista-Garrido J., Sun G., Obertas L., Mobley A.S., Kim G.S., Aronowski J, & Jung J.E. (2023). Young Astrocytic Mitochondria Attenuate the Elevated Level of CCL11 in the Aged Mice, Contributing to Cognitive Function Improvement. International Journal of Molecular Sciences, 24(6), 5187.

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Targeted siRNA Knockdown in Neural Progenitors

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siRNA-mediated knockdown was employed to downregulate cellular expression of BASP1, BASP1-AS1, and TCF12 in NPCs. Predesigned siRNA (Invitrogen) was used at a concentration of 40 pmoles. Cells were seeded at 80% confluency in 12-well format. According to the manufacturer’s protocol, knockdown was carried out using RNAimax (Invitrogen). For the control group, scrambled siRNA (Sigma) was used at a concentration of 40 pmoles. Transfection was carried out for 24 h, and samples were processed as per experimental requirement. To check the effect of BASP1-AS1-KD on the third day of differentiation, we performed knockdown on days 1 and 2 using siRNA.

Prajapati B., Fatima M., Fatma M., Maddhesiya P., Arora H., Naskar T., Devasenapathy S., Seth P, & Sinha S. (2020). Temporal transcriptome analysis of neuronal commitment reveals the preeminent role of the divergent lncRNA biotype and a critical candidate gene during differentiation. Cell Death Discovery, 6, 28.

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Silencing Regulatory Proteins via siRNA

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siRNA oligonucleotides for MVP, B7-H3, and MEK1/2 were purchased from Sigma with a scrambled siRNA (Sigma) serving as a control. Transfection was performed using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer’s protocol. Forty-eight hrs after transfection, the cell lysates were prepared for further analysis by Western blotting.

Liu Z., Zhang W., Phillips J.B., Arora R., McClellan S., Li J., Kim J.H., Sobol R.W, & Tan M. (2018). Immunoregulatory Protein B7-H3 Regulates Cancer Stem Cell Enrichment and Drug Resistance through MVP-mediated MEK Activation. Oncogene, 38(1), 88-102.

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Silencing Hes1 Gene Expression

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Small interfering RNA (siRNA) duplexes targeting human Hes1 sequences and a scrambled siRNA were purchased from Sigma-Aldrich. Transfection of the siRNA duplexes was performed by TransIT-TKO Transfection Reagent (Takara, Kusatsu, Shiga, Japan) according to the manufacturer’s instructions.

Saito N., Hirai N., Aoki K., Suzuki R., Fujita S., Nakayama H., Hayashi M., Ito K., Sakurai T, & Iwabuchi S. (2019). The Oncogene Addiction Switch from NOTCH to PI3K Requires Simultaneous Targeting of NOTCH and PI3K Pathway Inhibition in Glioblastoma. Cancers, 11(1), 121.

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