Synthetic shRNA targeting human GADD45b and non-targeting control siRNA (Invitrogen) were transfected into differentiated podocytes with Lipofectamine RNAi MAX reagent (Invitrogen, Carlsbad, CA). The target sequences of double-stranded nucleotides used for siRNA knockdown are 5′-ACGAGTCGGCCAAGTTGATGAATGT-3′ for GADD45B (1), 5′-CAGTCCTTCTGCTGTGACAACGACA-3′ for GADD45B (2), 5′-GAGGTGGCCAGCTACTGCGAAGAAA-3′ for GADD45B (3). A nonspecific, red fluorescence-labeled, double-strand RNA (BLOCK-iT Alexa Fluor Red Fluorescent Oligo (Invitrogen), was transfected in parallel as an siRNA-negative control. Synthetic shRNA transfection was performed according to the manufacturer's protocol. At 48 h after transfection, cells were treated with TGF-β and PAN for 24 h. The total protein extracts from the cells were used for western blot analysis.
Non targeting control sirna
Manufactured by Thermo Fisher Scientific
Sourced in United States
Non-targeting control siRNA is a laboratory reagent designed to serve as a control for RNA interference (RNAi) experiments. Its primary function is to provide a negative control to help evaluate the specific effects of target gene silencing in RNAi studies, without exhibiting any known biological activity or targeting a specific gene of interest.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (6 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions)
Lipofectamine transfection reagent, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Gene specific sirna, by Thermo Fisher Scientific (2 mentions)
Susd3, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Serbp1 sirna 1, by Thermo Fisher Scientific (2 mentions)
Serbp1 sirna 2, by Thermo Fisher Scientific (2 mentions)
Block it alexa fluor red fluorescent oligo, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 regent, by Thermo Fisher Scientific (1 mentions)
Sirna transfection reagent, by Thermo Fisher Scientific (1 mentions)
Transmessenger transfection reagent, by Qiagen (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Hiperfect, by Qiagen (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
P53 sirna, by Thermo Fisher Scientific (1 mentions)
Lmax microplate luminometer, by Molecular Devices (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
37 protocols using non targeting control sirna
1
Investigating GADD45b Knockdown in Podocytes
2
Silencing SBSN Gene Expression
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siSBSN was transfected into cells using Lipofectamine transfection reagent (Invitrogen, Tokyo, Japan) according to the manufacturer's instructions. The sequence of siSBSN was 5′-UAUUGAUGCCUUCAAGGGCCUUGCC-3′ (siSBSN1) and 5′-UUCCCUUCCAGCUUGAGUGAUUCCG-3′ (siSBSN2). A nontargeting control siRNA was used (Invitrogen).
3
Silencing H19 Modulates Chemosensitivity
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siRNA specific for H19 was purchased from Invitrogen (Thermo Fisher Scientific). To minimize nonspecific effects of interfering RNAs, non-targeting control siRNA, also obtained from Invitrogen, was used as negative control (NC). The siRNA transfection reagent (Invitrogen) was used according to the manufacturer’s protocol. Briefly, 3 × 104 cells in media without antibiotic were plated in 6-well plates and then transfected with 100 nM siRNA for H19 or NC when cells were 70% confluence the plate well at 24 hours using lipofectamine 2000 regent (Thermo Fisher Scientific). The cells were sampled at indicated time point for the different experiments with indicated purposes such as cell proliferation, invasion, and protein expression. For the validation of chemosensitivity experiment, cells with 24 hours siRNA transfection and then treated with tamoxifen in multiple concentrations and analyzed the cell proliferation and apoptosis rate.
4
PTEN Knockdown Via siRNA Transfection
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For transient knockdown of PTEN, cells were transfected with 150 pmol Stealth RNAi siRNA directed against PTEN, cFLIP (Invitrogen), or non-targeting control siRNA (Invitrogen) using the TransMessenger transfection reagent (Qiagen, Hilden, Germany).
5
Silencing GALNT2 in Neuroblastoma Cells
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Specific duplex si-RNA against GALNT2 and a non-targeting control si-RNA (Invitrogen, Life Technologies Inc.) were obtained for further experiments. SK-N-DZ cells were transfected for 48 hours with 20 nmol/L si-RNA using Lipofectamine RNAiMAX (Invitrogen, Life Technologies Inc.). For stable knockdown of GALNT2, SK-N-DZ cells were infected with shGALNT2/pLKO.1-puro (shGALNT2) or shCtrl/pLKO.1-puro (shCtrl; RNAi Core, Academia Sinica) using lentivirus-based infection system. After selected with 1 μg/mL puromycin (Millipore) for 2 weeks, cells were injected subcutaneously into mice for in vivo cell growth observation.
6
Transfection of Melanoma Cells with iNOS
7
Silencing Key Angiogenic Factors
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Hif1a siRNA, ANGPTL4 siRNA, Vegf siRNA, and a nontargeting control siRNA were purchased from Ambion. Predesigned control (scrambled [scr]), NRP1, and NRP2 siRNA sequences were obtained from Qiagen. siRNA delivery to cells was performed using Hiperfect (Qiagen).
8
Knockdown and Overexpression of Signaling Proteins
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siRNA transfections were performed as previously described [55 (link)] with 100ηM of either a non-targeting siRNA control or siRNA smart pools targeting p53, IKKβ, IKKα or KRAS (Dharmacon/Thermo Scientific, Pittsburgh, PA). For p53 knockdown studies in murine cells, we used 50ηM of p53 siRNA or non-targeting control siRNA (Ambion/Life Technologies, Grand Island, NY). Plasmid DNA transfection using a pcDNA-p53 expression vector (Addgene, Cambridge, MA) were performed with Lipofectamine LTX (Life technologies, Grand Island, NY) according to the manufacturer´s instructions. Combined siRNA/plasmid transfections were performed sequentially, with the plasmid transfection performed 48h after the siRNA transfection. Dual Luciferase Reporter assays were performed as described [55 (link)]. For pharmacological studies, cells were treated with CmpdA or vehicle control as indicated (see figure legends). Relative light units were measured on an Lmax Microplate Luminometer (Molecular Devices, Sunnyvale, CA).
9
Establishing Cell Lines for Pancreatic Cancer Research
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PANC-1 and PANC10.05 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). BxPC-3, MiaPaca-2, CFPAC-1, Colo357 and Capan-1 cell lines were provided by Dr Joerg Hoheisel and HPNE and HPDE cell lines were provided by Dr Christoph Roesli (both DKFZ, Heidelberg). HEK293FT cells were obtained from Invitrogen (Carlsbad, CA, USA). All cell lines used were tested for Mycoplasma infection and were verified for authenticity (DKFZ core facility). PANC10.05 cells stably overexpressing miR-206 were generated using the pBABE-puro retroviral system. The pre-miR-206 sequence along with 300 nucleotide-long flanking sequence was cloned into pBABE-puro vector using BamHI and XhoI restriction enzymes. PANC10.05 cells were transduced with pBABE-empty (control) or pBABE-miR-206 and selected in 2 mg/ml puromycin (Invitrogen).
Transfections were performed using using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. All siRNAs, as well as the non-targeting control siRNA, were from Ambion (Austin, TX, USA). The second siRNA against ANXA2 gene (siANXA2#2) was from Dharmacon (Lafayette, CO, USA). For each gene, 2 individual siRNAs were used (Supplementary Table S6 ). miRIDIAN miRNA mimics and the negative control were obtained from Dharmacon. siRNAs and miRNA mimics were used at a final concentration of 30 nM , unless mentioned differently.
Transfections were performed using using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. All siRNAs, as well as the non-targeting control siRNA, were from Ambion (Austin, TX, USA). The second siRNA against ANXA2 gene (siANXA2#2) was from Dharmacon (Lafayette, CO, USA). For each gene, 2 individual siRNAs were used (
10
Silencing Caspase Genes by siRNA
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RNA interference was done as described
previously.45 (link) Briefly, siRNA for caspase-8,
-9, and -3 was purchased from Dhamacon Research, Inc. Nontargeting
control siRNA was purchased from Ambion. Transfections were performed
using Lipofectamine RNAiMAX (Invitrogen) in the reverse manner according
to the manufacturer’s instructions. Between 5 and 10 pmol of
siRNA and 5 μL of Lipofectamine RNAiMAX were mixed in each well
of 6-well plates for 20 min, followed by culturing 3 × 105 cells in the siRNA mix for 24–48 h; knockdown efficacy
was assessed by Western blotting.
previously.45 (link) Briefly, siRNA for caspase-8,
-9, and -3 was purchased from Dhamacon Research, Inc. Nontargeting
control siRNA was purchased from Ambion. Transfections were performed
using Lipofectamine RNAiMAX (Invitrogen) in the reverse manner according
to the manufacturer’s instructions. Between 5 and 10 pmol of
siRNA and 5 μL of Lipofectamine RNAiMAX were mixed in each well
of 6-well plates for 20 min, followed by culturing 3 × 105 cells in the siRNA mix for 24–48 h; knockdown efficacy
was assessed by Western blotting.
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