Lipofectamine 3000 (Invitrogen, USA) was used as the reagent for cell transfection as the manufacturer’s instruction describes. Briefly, cultured cells were cultured to ~ 80% confluence. The short hairpin RNAs (shRNAs) targeting SPHK1, SIRT1 and FoxO3, miR-19a/b-3p mimics, miR-19a/b-3p inhibitor, and corresponding negative controls were obtained from GenePharma (Shanghai, China). Corresponding constructs above together with Lipofectamine 3000 (m/v 1:1) were directly added into the culture media for 48 h followed by harvest.
Short hairpin rnas shrnas
Manufactured by GenePharma
Sourced in China
Short hairpin RNAs (shRNAs) are a type of laboratory equipment used in molecular biology research. shRNAs are small, double-stranded RNA molecules designed to induce gene silencing through the RNA interference (RNAi) pathway. They function by targeting specific messenger RNA (mRNA) molecules, leading to their degradation or translational repression, thereby reducing the expression of the corresponding gene.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Hct116, by Thermo Fisher Scientific (1 mentions)
Recombinant lentiviral vector, by Genechem (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Sw620, by American Type Culture Collection (1 mentions)
Sw480, by Thermo Fisher Scientific (1 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (1 mentions)
Sw620, by Thermo Fisher Scientific (1 mentions)
Ncm460, by Thermo Fisher Scientific (1 mentions)
Htc116, by American Type Culture Collection (1 mentions)
Mimic nc, by GenePharma (1 mentions)
Mir 134 5p mimic, by GenePharma (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Anti e cadherin, by BD (1 mentions)
9 protocols using short hairpin rnas shrnas
1
Cell Transfection Optimization
2
Colorectal Cancer Cell Culturing and Genetic Manipulation
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Human colon mucosal epithelial cells NCM460 and different human colorectal tumor cell lines (LoVo, HTC116, SW480, T84, SW620) were purchased from ATCC (Manassas, USA) and cultured in a 5% CO2/37 °C incubator. NCM460, HCT116, and T84 were maintained in McCoy’s 5A medium (Invitrogen, Carlsbad, USA), whereas LoVo, SW480, and SW620 cells were cultured in RPMI-1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS). Recombinant lentiviral vectors carrying FBXW11 or HIC1, and corresponding control vectors (named Control and mock, respectively) were designed and generated by GeneChem (Shanghai, China). Short hairpin RNAs (shRNAs) targeting FBXW11 (shFBXW11-1 and shFBXW11-2), SIRT1 (shSIRT1), and HIC1 (shHIC1), as well as corresponding scrambled controls (named Scramble and shRNA, respectively) were synthesized by GenePharma (Shanghai, China). Lipofectamine® 2000 Transfection Reagent (Invitrogen) was used to transfect colorectal tumor cell cells with designated lentiviral vectors and/or shRNAs. Transfection efficiency was evaluated by qRT-PCR and/or western blotting prior to further analyses.
3
Knockdown of lnc-CYB561-5 in Lung Cancer Cells
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Short hairpin RNAs (shRNAs) were designed and synthesized by GenePharma. The shRNA‐ctrl vector and shRNA‐lnc‐CYB561‐5 were transfected into H1299 and H292 cells using Lipofectamine 2000 transfection reagent (Invitrogen). Recombinant lentivirus (Bsg‐LV, Vector‐LV) were prepared by Genechem Co., Ltd. The titre of the concentrated virus suspension was 5 × 1011 Tu/L. Cells were infected with lentivirus for 48 h and then selected with 2 ng/ml puromycin for 2 weeks, with the medium refreshed every 3 days.
4
Modulating SMAD6 and miR-134-5p in Rb Cells
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The short hairpin RNAs (shRNAs) targeting SMAD6 or DNM3OS were synthesized by GenePharma (Shanghai, China). The miR-134-5p mimics and NC-mimics were also manufactured by GenePharma. The pcDNA3.1 vectors for overexpression of SMAD6 were provided by GeneChem (Shanghai, China). The aforementioned plasmids were introduced into Weri-Rb1 andY79 cells using lipofectamine 3000 reagent (Invitrogen).
5
Stable Knockdown of HOTAIRM1, FUS, and E2F7
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Short hairpin RNAs (shRNAs) targeting HOTAIRM1, FUS, and E2F7 were all provided by GenePharma (Shanghai, China). For cell transfection, serum-free medium (500 μl), plasmid (4 μg), and lipofectamine 3000 (10 μl, Invitrogen, CA, USA) were mixed and left at room temperature for 20 min to form the RNA/lipo mixture. Subsequently, the mixture was added to the serum-free medium cultured cells. After 6 h, the used medium was replaced with fresh complete medium, and the culture was continued for 48 h. For the construction of stable transfected cell lines, virus solution (100 μl) was added to complete medium (1 ml), and then polybrene with a concentration of 10 mg/ml (0.5 μl) was added and mix (final polybrene concentration is 5 μg/ml). When the cell density is about 80%, the used medium is discarded, virus infection mixture (0.25 ml) is added to each well of the 24-well plate. After 24 h of incubation, the virus infection mixture was discarded, and fresh complete medium was added to continue the culture for 24 h. Cells were passaged when the cells grew to 90% density. After the cells adhered, complete medium containing puromycin (2 μg/ml) was added to each well for selection.
6
Comprehensive Chromatin Remodeling Antibody Panel
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The sources of the antibodies were anti-FLAG, anti-HDAC1 anti-HDAC2, anti-RbAp46/48, anti-Fibronectin, anti-Vimentin and anti-β-actin (Sigma–Aldrich); anti-DDB1, anti-MTA1, anti-SIN3A, anti-SAP180 and anti-SAP30 (Santa Cruz Biotechnology); anti-PRMT5, anti-ROC1, anti-LSD1, anti-DNMT3B, anti-HDAC5 and anti-MTA2 (Abcam); anti-EED, anti-MBD2/3 and anti-MTA3 (Millipore); anti-SUZ12 (Cell Signaling Technology); anti-WDR77 (also known as MEP50, Bethyl); anti-MTA2, anti-E-cadherin, anti-α-catenin, anti-γ-catenin, anti-N-cadherin and anti-EZH2 (BD Bioscience); anti-ING4 (Genetex), anti-SETD2 (Proteintech). Dynabeads Protein G was obtained from Invitrogen by Thermo Fisher Scientific, and protease inhibitor mixture cocktail was from Roche Applied Science. Glutathione-Sepharose 4B beads were from GE Healthcare Bio-Sciences. Short hairpin RNAs (shRNAs) were obtained from GenePharma Co Ltd (Shanghai, China).
7
Silencing PUM2 and SIRT1 in Neurons
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Short hairpin RNAs (shRNAs) targeting PUM2 (shPUM2), SIRT1 (shSITR1) and respective shNCs were obtained from GenePharma. Lipofectamine 3000 transfection reagent (Invitrogen) was employed for Lentiviral (LV) transduction or shRNA transfection into the mice or cortical neurons.
8
Investigating PLK2, SKP1, and α-Syn
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Short hairpin RNAs (shRNAs) for PLK2 and SKP1, overexpression plasmids for PLK2 and α-Syn (pcDNA3.1-α-Syn), and corresponding negative controls were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). miR-101-3p mimic, inhibitor, and negative control were purchased from RiboBio (Guangzhou, China). Transfection was conducted according to the instruction of the manufacturer.
9
Overexpression and Knockdown of NOL4L and CASC11
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The forced expression of NOL4L was achieved by constructing PCR-amplified NOL4L sequence into pcDNA3.1 vector (Invitrogen) to establish pcDNA3.1/NOL4L overexpression plasmids. Short hairpin RNAs (shRNAs) against CASC11 (sh/CASC11#1/2/3) and controls sh/NC, miR-676-3p mimics and miR-676-3p inhibitor and their controls NC mimics, and NC inhibitor were attained from GenePharma Co. Ltd (Suzhou, China). Abiding by the protocols of suppliers, we transfected these plasmids into SK-N-AS and NB-1 cells using Lipofectamine 2000 reagent (Invitrogen).
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