HOTTIP short hairpin (sh)RNA (sh-HOTTIP) or negative controls (sh-NC) were obtained from Shanghai GenePharma Co., Ltd. A pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) was used for the construction of the overexpression plasmid pcDNA3.1-HOTTIP. The HOTTIP sequence (RefSeq, NR_037843.3) was purchased from Sangon Biotech Co., Ltd. and subcloned into the pcDNA3.1 vector; an empty pcDNA 3.1 vector was used as the control.
Pcdna3.1 vector
Manufactured by Sangon
Sourced in China
PcDNA3.1 vector is a plasmid commonly used in molecular biology experiments. It functions as a cloning and expression vector, enabling the insertion and expression of foreign DNA sequences in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (18 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (12 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (3 mentions)
Si pten, by Sangon (3 mentions)
Pcdna3.1 vector, by Vector Laboratories (3 mentions)
Mir nc, by GenePharma (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Si nc, by Sangon (2 mentions)
Si nc, by GenePharma (2 mentions)
Dual luciferase assay system, by Promega (2 mentions)
Prl sv40 renilla luciferase plasmid, by Promega (2 mentions)
Pischeck 2 vector, by Promega (2 mentions)
Pgl3 basic, by Promega (2 mentions)
Lipofectamine 2000, by Merck Group (2 mentions)
Ht1197, by American Type Culture Collection (2 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Sh nc, by GenePharma (1 mentions)
Small interfering rnas sirna, by GenePharma (1 mentions)
62 protocols using pcdna3.1 vector
1
HOTTIP Overexpression and Knockdown
2
GAS5 Gene Modulation in Osteosarcoma Cells
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Human GAS5 gene was ampli ed by PCR from hFOB1.19 cells and the cDNA sequences were subcloned into pcDNA3.1 vector (Sangon, Shanghai, China) to construct pcDNA-GAS5 plasmid, with the empty pcDNA3.1 vector serving as the negative control (Vector). Plasmids were transfected into U2OS and HOS cells using Lipofectamine 3000 Transfection Reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). MiR-23a-3p mimic, inhibitor, and its corresponding negative control mimic or inhibitor (NC-mimic and NC-inhibitor) were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In addition, siRNA negative control (Scramble), si-GAS5, and si-PTEN (All obtained from Sangon, Shanghai, China) were transfected into cells as described above.
3
Molecular Cloning of lncNSPL, RIG-I, and TRIM25 Constructs
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Sequence of lncNSPL (Gene number: 105370355,accession: XR_007083671), RIG-I and TRIM25 genes were obtained from the NCBI database. The lncNSPL plasmid construction process is divided into two steps. In the first step, the full length of lncNSPL was synthesized and cloned into pUC57 vector (Sangon Biotech, Shanghai, China). And then the full-length lncNSPL was cloned into the pcDNA3.1 eukaryotic expression vector and the retroviral vector GV367 by molecular cloning techniques, respectively. In this retroviral vector construct, lncNSPL transcribed was from RNA Pol II promoter of retroviral vectors GV367. Coding sequences of RIG-I and TRIM25 were cloned into pCDNA3.1 vector containing HA and Flag tags, respectively (Sangon Biotech, Shanghai). HA-tagged truncated structures of RIG-I were generated by standard molecular biology methods. Myc-Ub-K63 structure was a gift from Dr. Q. Zhang (Zhejiang University, China). Full-length lncNSPL was cloned in retroviral vectors GV367 for stable expression in THP-1 cells. LV10 lentiviral vector expressing control shRNA against RFP or shRNA targeting lncNSPL were obtained from Gene Pharma (Shanghai, China).
Small interfering RNAs (siRNAs) were designed and synthesizeds by GenePharma (Shanghai, China). The transfection of plasmids or siRNAs were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions.
Small interfering RNAs (siRNAs) were designed and synthesizeds by GenePharma (Shanghai, China). The transfection of plasmids or siRNAs were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions.
4
Overexpression of snaR in Liver Cancer Cells
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HEP G2 and C33A were provided by American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured with RPMI-1640 medium (ATCC) containing 10% fetal bovine serum (FBS; ATCC) at 37°C in an incubator with atmosphere of 5% CO2. Full-length snaR was reverse transcribed into cDNA using the aforementioned method. snaR was amplified and inserted into a pcDNA3.1 vector (Sangon Biotech Co., Ltd., Shanghai, China) to make an snaR expression vector. The vectors were transfected into cells at a dose of 50 nM using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Expression of snaR was detected by RT-qPCR. Subsequent experiments were performed 24 h after transfection only in cases where snaR overexpression was increased >200% compared with the control untransfected cells and mock-transfected negative control cells (cells transfected with empty vector). For treatment with exogenous TGF-β1 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and TGF-β inhibitor SD 208 (R&D Systems China Co., Ltd., Shanghai, China), cells (105 cells/ml) were treated for 24 h at 37°C prior to use. TGF-β1 was used at concentrations of 10 and 30 ng/ml and TGF-β inhibitor was used at a dose of 10 ng/ml, based on the manufacturers' protocols.
5
Transfection of CSCC Cell Lines
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C33A and SiHa human CSCC cell lines (ATCC, USA) were used as CSCC cell model. Cell culture medium contained 10% FBS and 90% Eagle’s Minimum Essential Medium. Cells were cultivated in a 5% CO2 incubator at 37 °C with 95% humidity. Cells were harvested at 70–80% confluence to perform subsequent transfections.
Vectors expressing miR503HG and Caspase-3 were constructed using the pcDNA3.1 vector (Sangon, Shanghai, China). Negative control (NC) miRNA and miR-155 mimic were the products of Sangon (Shanghai, China). Lipofectamine 2000 (Sangon) was used to transfect 10 nM vectors (empty vector as NC group) or 50 nM miRNA (NC miRNA as NC group) into 106 cells. All operations were completed according to the manufacturer’s instructions. All the following experiments were performed at 24h post-transfection.
Vectors expressing miR503HG and Caspase-3 were constructed using the pcDNA3.1 vector (Sangon, Shanghai, China). Negative control (NC) miRNA and miR-155 mimic were the products of Sangon (Shanghai, China). Lipofectamine 2000 (Sangon) was used to transfect 10 nM vectors (empty vector as NC group) or 50 nM miRNA (NC miRNA as NC group) into 106 cells. All operations were completed according to the manufacturer’s instructions. All the following experiments were performed at 24h post-transfection.
6
Prostate Cancer Cell Line Manipulation
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VCaP and LNCaP cell lines were cultivated all night to reach 70–80% confluence. All cell transfections were operated by use of lipofectamine2000 (Thermo Fisher Scientific). The pcDNA3.1 vector expressing PAX5, PTBP3, or ATG5 and empty vector (named as Ctrl) were designed and constructed by Sangon (Shanghai, China). To knockdown the expression of PAX5 or IDH1-AS1, the short hairpin RNAs (shRNAs) against PAX5 or IDH1-AS1 (named as shPAX5 or shIDH1-AS1#1/2) and non-specific shRNA (named as shCtrl) were synthesized by GenePharma (Shanghai, China). MISSION® microRNA Mimics miR-216b-5p and scrambled negative control (named as miR-NC) were from Sigma-Aldrich (St. Louis, MO, USA). After 48 h of incubation, cells were reaped from three independent experiments and used for subsequent analysis.
7
miR-135b-5p Modulation in GSK-3β Regulation
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miR-135b-5p mimics, miR-135b-5p inhibitor, and negative control (NC) were purchased from Ambion (Austin, TX, USA). The pcDNA3.1/GSK-3β vector was generated by inserting the open reading frame of GSK-3β without its 3′-UTR into the pcDNA3.1 vector (Sangon Biotech, Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions.
8
miR-590-5p Regulation of STAT3 Expression
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Cells were transfected with miR-590-5p mimics, inhibitor, or negative control (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA fragment of the STAT3 open reading frame was inserted into the pcDNA3.1 vector (Sangon Biotech, Shanghai, China). This construct was transfected into cells using Lipofectamine 2000 (Invitrogen). After 48 h from transfection, cells were treated with 25 ng/ml PDGF (R&D Systems, Minneapolis, MN, USA) and incubated for 24 h.
9
Modulating HCG11, miR-579-3p, and MDM2 Expression
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Small fragments including si-HCG11-1, si-HCG11-2, si-RNA negative control (NC), pcDNA3.1-vector, pcDNA3.1-HCG11, pcDNA3.1-MDM2, miR-579-3p mimic and NC were designed and synthesized by Sangon (Shanghai, China). Then, the fragments were transfected into cells by using Lipofectamine 2000 to regulate the expression of HCG11, miR-579-3p and MDM2, respectively.
10
Transient Transfection of CIHP-1 Cells
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The CASC15 expression vector was constructed using pcDNA3.1 vector (Sangon, Shanghai, China). Negative control (NC) miRNA and miR-34c mimic were from Sangon. CIHP-1 cells were counted. The transient transfections were performed using lipofectamine 2000 reagent (Invitrogen, USA) to transfect 15 nM vectors (empty vector as NC group) or 35 nM miRNAs (NC miRNA as NC group) into 2×106 cells. The following experiments were performed using cells harvested at 24 h after transfection. For all transfections, control (C) cells were untransfected cells.
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