Scrambled shrna

Manufactured by Thermo Fisher Scientific

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Scrambled shRNA is a laboratory tool used in RNA interference (RNAi) experiments. It consists of a short hairpin RNA (shRNA) sequence that does not target any known gene, serving as a negative control for RNAi studies. Scrambled shRNA allows researchers to assess the off-target effects and background levels of RNAi in their experiments.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions) C56bl 6 lck cre mice, by Taconic Biosciences (1 mentions) Zeocin, by Thermo Fisher Scientific (1 mentions) Scrambled shrna, by Genechem (1 mentions) Scrambled shrna, by GenePharma (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions)

5 protocols using scrambled shrna

Generating Rictor Knockout Mice

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Homozygous C56BL/6 rictor^f/f mice (14 (link)) were crossed with C56BL/6 Lck-Cre mice
(Taconic farms, NY), which generates T-cell-specific rictorknockout mice (rictor^T−/−) owing to the expression of
Cre under the control of the proximal promoter of Lck. To specifically delete
rictor in the OT-1 TCR background, we crossed
C56BL/6/OT-1/β2m^−/-/Rag-2^−/-mice (12 (link)) with homozygous
rictor^T−/− to obtain
OT-1/Rag-2^−/-/rictor^T−/−animals (referred to as OT-1/rictor^T−/−). All mice
were genotyped by PCR using the respective primers described in Supplemental Table 1.
Handling and experimentation protocols have been reviewed and used in accordance
with IACUC regulations of Rutgers University. Tissues were removed from
6-week-old rictor^T−/− and rictor^T+/+littermates, micro-sliced and resuspended in RIPA buffer (50 mM Tris-HCl pH8.0,
100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.2% SDS, 0.5%
Na-deoxycholate supplemented with protease inhibitors). Jurkat cells were
electroporated and transfected with rictor shRNA or scrambled shRNA (Invitrogen,
CA). Transfected cells were cultured in complete RPMI medium in the presence of
the selection antibiotic Zeocin (Invitrogen, CA). SIN1^−/-MEFs cells were cultured as previously described (15 (link)).

Chou P.C., Oh W.J., Wu C.C., Moloughney J., Rüegg M., Hall M.N., Jacinto E, & Werlen G. (2014). mTOR complex 2 modulates αβT-cell receptor processing and surface expression during thymocyte development. Journal of immunology (Baltimore, Md. : 1950), 193(3), 1162-1170.

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Podocyte Knockdown Assay for HG

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Gp91^phox shRNA and TXNIP shRNA were purchased from Genechem (Shanghai, China); meanwhile the scrambled shRNA (Genechem, Shanghai, China) was used as a control. Podocytes were transiently transfected with gp91^phox/TXNIP shRNA or scrambled shRNA by lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's instruction. Two days later, the podocytes were exposed to HG (30 mmol/L) for indicated times.

Gao P., He F.F., Tang H., Lei C.T., Chen S., Meng X.F., Su H, & Zhang C. (2015). NADPH Oxidase-Induced NALP3 Inflammasome Activation Is Driven by Thioredoxin-Interacting Protein Which Contributes to Podocyte Injury in Hyperglycemia. Journal of Diabetes Research, 2015, 504761.

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TRIM28 Knockdown using shRNA

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For TRIM28 knockdown, two kind of human TRIM28-targeting short hairpin RNA (shRNA) oligonucleotide sequences and a scrambled shRNA as a negative control were cloned into vector. The shRNA sequences were shTRIM28#1: CT-GAGACCAAA CCTGTGCTTA and shTRIM28#2: CCTG-GCTCTGTTCTCTGTCCT (Su et al. 2018) (link). The synthetic TRIM28-targeting shRNA, scrambled shRNA and TRIM28 mimics were obtained from GenePharma (Shanghai, China). After cells were cultured in 12-well plates for 24 h, TRIM28targeting shRNA, scrambled shRNA and TRIM28 mimics were transfected into cells by Lipofectamine 3000 (Invitrogen).

Chen Y., Cheng H, & Long H. (2021). Tripartite motif containing 28 (TRIM28) promotes the growth and migration of endometrial carcinoma cells by regulating the AKT/mTOR signaling pathway. General physiology and biophysics, 40(3).

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Knockdown of hepatic targets

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Cells were plated at a density of 200,000 cells per well of a six-well plate, 24 h prior to transfection. Transfection of Hep3B and Huh7 cells was performed using Lipofectamine™ 2000 (Thermo Fisher Scientific), in serum- and antibiotic-free OptiMem™ I Reduced Serum medium (Thermo Fisher Scientific), according to manufacturer’s instructions. Plasmids containing short hairpin RNA (shRNA) molecules directed against human TUBB2B, HNF4A, and CYP27A1 mRNA, or a scrambled shRNA (Thermo Fisher Scientific) were used. At 24, 48 and 72 h post-transfection, the cells were collected for Western blot analysis. Sequences of the shRNA used are shown in Supplementary file 3.

Wang X., Shi J., Huang M., Chen J., Dan J., Tang Y., Guo Z., He X, & Zhao Q. (2023). TUBB2B facilitates progression of hepatocellular carcinoma by regulating cholesterol metabolism through targeting HNF4A/CYP27A1. Cell Death & Disease, 14(3), 179.

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Modulating PKCδ in Neuroblastoma Cells

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All cell lines were maintained at 37°C in an incubator containing 5% CO₂. Human neuroblastoma SY5Y-APP741 and mouse neuroblastoma N2a-APP695 were cultured in 50% DMEM (HyClone) mixed with 50% OptiMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco), in the presence of 400 µg/ml G418 (Sigma-Aldrich).
The target sequence for human PKCδ for siRNA oligonucleotide duplexes was 5′-CCGCTTCAAGGTTCACAACTA-3′, and human PKCδ and control siRNA were both purchased from QIAGEN. shRNA for mouse PKCδ comprised a hairpin targeting 5′-CAGAGTTCCTGAATGAGAA-3′, and mouse PKCδ shRNA, scrambled shRNA, PKCδ plasmid control, and PKCδ plasmid WT were all purchased from Thermo Fisher Scientific. Cells were transfected with PKCδ siRNA or plasmid WT by using Lipofectamine 2000 (Invitrogen).

Du Y., Zhao Y., Li C., Zheng Q., Tian J., Li Z., Huang T.Y., Zhang W, & Xu H. (2018). Inhibition of PKCδ reduces amyloid-β levels and reverses Alzheimer disease phenotypes. The Journal of Experimental Medicine, 215(6), 1665-1677.

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