For transient miRNA transfection experiments, 100 nM of the miRNA mimics for hsa-miR-200b-3p and the negative miRNA mimic transfection control were obtained from Invitrogen and were reverse transfected at the time of seeding using Lipofectamine RNAimax (Life Technologies). Transient overexpression of PKCζ-WT and PKCζ-Kinase death was achieved by transfection using X-tremeGENE HP transfection reagent (Roche). Transfected cells were examined 48 h after transfection. To knock down Sfrp1, Sfrp2, Prrx1 and Prrx2, small interfering RNAs (siRNAs) for Sfrp1 (sc-39999), Sfrp2 (sc-40001), Prrx1 (sc-152531), Prrx2 (sc-152532) and nontargeting siRNA (negative-siRNA) were purchased from Santa Cruz Biotechnology. Pooled siRNAs were reverse transfected at the time of seeding according to the manufacturer’s protocol. To knock out Sox2, Sfrp1 and Sfrp2 in colonic fibroblasts, sgRNA targeting Sox2 exon1 (5’-CUCCAUCAUGUUAUACAUGC-3’), sgRNA targeting Sfrp1 exon1 (5’-UCGCCGAGCAACAUGGGCGU-3’), sgRNA targeting Sfrp2 exon2 (5’-UUCACACACCUUGGGAGCUG -3’) were purchased from Synthego and transduced into cells with recombinant Streptococcus pyogenes Cas9 protein (Truecut Cas9 Protein v2, Thermo). Sox2, Sfrp1 and Sfrp2 were knocked out using the Neon Transfection System 1 (Thermo) following the manufacturer’s protocol and single clones were expanded and screened by protein immunoblotting.
Negative sirna
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Negative siRNA is a laboratory tool used to study gene function. It is a synthetic small interfering RNA (siRNA) designed to target and silence a specific gene in a controlled manner, without affecting the expression of other genes. Negative siRNA serves as a negative control in experiments involving RNA interference (RNAi) techniques, allowing researchers to differentiate the effects of the target gene knockdown from other potential confounding factors.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Truecut cas9 protein v2, by Thermo Fisher Scientific (1 mentions)
X tremegene hp transfection reagent, by Roche (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Caki 1, by American Type Culture Collection (1 mentions)
Crl 1573, by American Type Culture Collection (1 mentions)
Htb 46, by American Type Culture Collection (1 mentions)
Crl 1611, by American Type Culture Collection (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Akt sirna, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Propofol, by Merck Group (1 mentions)
Hif1α small interfering rna sirna, by Santa Cruz Biotechnology (1 mentions)
5 protocols using negative sirna
1
Transient Modulation of miRNA and Protein Targets
2
Silencing TGM2 and HDM2 in Renal Cell Carcinoma
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Cell lines including ACHN, CAKI-1 and HEK293 were obtained from American Type Culture Collection (CRL1611, HTB-46, CRL1573 respectively; ATCC, Manassas, VA, USA). ACHN-luciferase cell line was obtained from Caliper Life Sciences (125056, Hopkinton, MA, USA). Cells were cultured in RPMI 1640 containing 10% fetal bovine serum under 5% CO2 and 100% humidity at 37 °C. A siRNA duplex targeting human TGM2 (siTGase 2; #10620318, #10620319, Invitrogen) and HDM2 was treated to cells for 48 h using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. As negative controls, cells were incubated with Lipofectamine RNAiMAX (Invitrogen) and a negative siRNA (Santa Cruz). Then, cells were left untreated or were treated with 50 μM of chloroquine to inhibit autophagolysosome fusion or 10 μM of MG132 to prevent proteasome degradation in amino acid-free Earle's balanced salts solution (EBSS; Sigma-Aldrich) for 6 h. In this study, siRNA was the mix of three individual siRNA duplexes per target gene designed using Rosetta Inpharmatics′ algorithm from company. As reviewer suggested a proof of the same effect using at least two independent siRNA, we have repeated experiments with two different siRNA sequences against targets of TGase 2, HDM2 and p53, which resulted in the same effect as the mix of three siRNAs (Supplementary Figure 2 ).
3
Silencing ClC-5 in Human Cell Lines
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The human normal osteoblasts hFOB1.19, osteosarcoma cell lines U-2OS, SaoS-2 and HOS, and human renal proximal tubule epithelial HK-2 cells were purchased from China Center Type Culture Collection (CCTCC, Shanghai, China). Cells were cultured in RPMI1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. The sequence of the stealth siRNA duplex oligoribonucleotides against human ClC-5 gene (5’-GCACTTCCATCATTCATTT-3’) and negative siRNA were synthesized and purchased from Santa Cruz Biotechnology. The siRNAs were transfected transiently with Lipo RNAi max Reagent (Applied Biosystems, CA, USA) for 48 h according to the manufacturer’s instructions.
4
Knockdown of SRPK2 and Akt in Microglia
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The cells were seeded (1 × 105 cells/well) into 12-well culture plates and transfected with 40 nM of SRPK2 siRNA, Akt siRNA (Santa Cruz Biotechnology, Dallas, TX, USA), or negative siRNA (Santa Cruz Biotechnology) using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific) according to the manufacturer's instructions. At 24 h post-transfection, western blotting was performed to assess transfection efficiency, and the cells were used for subsequent experiments. The BV2 cells with SRPK2 knockdown were then stimulated by Aβ or LPS + IFN-γ, while the BV2 cells with Akt knockdown were stimulated by Aβ.
5
Prostate Cancer Cell Culture Conditions
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The human prostate cancer cell lines PC3, DU145, and 22RV1 were purchased from American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). All cells were incubated at 37°C in a humidified atmosphere containing 21% O2 and 5% CO2. For hypoxic culture, the cells were placed in a hypoxic incubator (1% O2, 5% CO2) at 37°C for 6 h. HIF-1α small interfering RNA (siRNA) and negative siRNA were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Propofol was purchased from Sigma-Aldrich.
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