For circ-NRIP1 downregulation, small interfering RNA (siRNA) against circ-NRIP1 (si-circ-NRIP1) and its negative control (si-NC) was constructed by Genepharma (Shanghai, China). For circ-NRIP1 upregulation, circ-NRIP1 sequence was amplified by PCR and inserted into pcDNA vector (Invitrogen, Carlsbad, CA, USA) to generate fusion plasmids, named as circ-NRIP1, pcDNA empty vector (vector) as the control. For miR-186-5p enrichment or inhibition, miR-186-5p mimics (miR-186-5p), miR-186-5p inhibitor (anti-miR-186-5p) and its own negative control (miR-NC and anti-miR-NC) were purchased from Ribobio (Guangzhou, China). For MYH9 overexpression, MYH9 sequence was amplified by PCR and inserted into pcDNA vector (Invitrogen) to generate fusion plasmids, namely MYH9, pcDNA empty vector (vector) as the control. All items were introduced into HGC-27 and AGS cells using Lipofectamine 2000 (Invitrogen). At 48 h post-transfection, HGC-27 and AGS cells were collected and used for further analyses. The used sequence was listed as below: si-circ-NRIP1 sequence (3ʹ-CTAGGGAATCCAAACGTTCA-5ʹ).
Pcdna vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The PcDNA vector is a plasmid-based expression system designed for the efficient expression of recombinant proteins in mammalian cell lines. It contains a strong cytomegalovirus (CMV) promoter to drive high-level expression of the gene of interest, as well as a selectable marker for stable cell line generation.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (11 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (10 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (6 mentions)
Si nc, by GenePharma (5 mentions)
Mir nc, by RiboBio (5 mentions)
Si nc, by RiboBio (4 mentions)
Pcdna empty vector, by Thermo Fisher Scientific (3 mentions)
Anti mir nc, by RiboBio (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Si uca1 1, by GenePharma (2 mentions)
Si uca1 2, by GenePharma (2 mentions)
Si uca1 3, by GenePharma (2 mentions)
Mir 143 mimic mir 143, by RiboBio (2 mentions)
Mir nc, by GenePharma (2 mentions)
Anti mir nc, by GenePharma (2 mentions)
Fetal bovine serum (fbs), by Beyotime (2 mentions)
Mir 186 5p, by RiboBio (1 mentions)
Anti mir 186 5p, by RiboBio (1 mentions)
Plko 1 vector, by Addgene (1 mentions)
Polybrene, by Merck Group (1 mentions)
42 protocols using pcdna vector
1
Modulating circ-NRIP1 and miR-186-5p in Gastric Cancer Cells
2
Establishing Circular RNA and ITGB1 Constructs
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Full-length sequences of circ_0000735 and integrin-β1 (ITGB1) were, respectively, cloned into the empty pCD5-ciR vector (Geneseed, Guangzhou, China) and pcDNA vector (Thermo Fisher, Waltham, MA, USA) to establish pCD5-ciR-circ_0000735 (circ_0000735) and pcDNA-ITGB1 (ITGB1) plasmids. All oligonucleotides were synthesized by Sangon Biotech Co., Ltd (Shanghai, China), including a siRNA targeting circ_0000735 (si-circ_0000735), miR-153-3p inhibitor (anti-miR-153-3p), miR-153-3p mimic (miR-153-3p), and their negative controls si-NC, anti-miR-NC, and miR-NC. Transfection of the cells with oligonucleotides and/or plasmids was executed with Lipofectamine 3000 (Thermo Fisher). HEK-293T cells were transfected with the recombinant pLKO.1 vector (Addgene, Cambridge, MA, USA) carrying sh-circ_0000735 or sh-NC (1 μg) along with psPAX2 packaging plasmid (750 ng) and pMD2.G envelope plasmid (250 ng). Lentivirus particles from HEK-293T cells were collected and then used to infect A549 cells under polybrene (8 µg/mL, Sigma).
3
Manipulating AEG-1 and let-7d Expression
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pcDNA-AEG-1 plasmid was established by inserting the full-length sequence of AEG-1 into pcDNA vector (Thermo Fisher Scientific). To manipulate the expression of AEG-1 and let-7d, IOMM-Lee and CH-157MN cells were transfected with let-7d mimics (GenePharma, Shanghai, China), let-7d inhibitor (GenePharma) or pcDNA-AEG-1 using Lipofectamine 2000 (Thermo Fisher Scientific). Scramble RNA (miR-control) and pcDNA empty vector were used as negative controls.
4
Establishing Stable Cell Lines for EK and RIG-I Knockdown
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To establish stable 293T clones expressing EK or EK-X2, we transfected the cells with a pcDNA vector (Thermo Fisher) expressing the EK or EK-X2 cDNA together with a hygromycin-resistance gene. After selection with 150 μg/ml hygromycin B (Thermo Fisher), several independent clones expressing EK or EK-X2 were isolated and analyzed.
To establish stable A549 cells, we used a lentiviral vector (the pLVX series, TaKaRa) to express small hairpin RNAs (shRNAs) under the control of the U6 promoter (the target sequences knocked down are listed in Table3 ), together with a puromycin-resistance gene or blasticidin-resistance gene under the control of the PGK promoter. The recombinant lentiviruses were constructed by simultaneously transfecting 293T cells with the pLVX vector and lentiviral plasmids expressing Gag/Pol, Rev, and VSVG (Naldini et al., 1996 (link)). After selection with 1.3 μg/ml puromycin (Nacalai Tesque, Inc., Kyoto, Japan), the puromycin-resistant A549 cells expressing RIG-I-targeting shRNA were pooled, and stored for analysis and subsequent experiments. The sh-RIG-I A549 cells were then infected with recombinant lentiviruses expressing shRNAs targeting different sites in the EK mRNA (sh-EK#1, sh-EK#2, and control shRNA), and selected with 180 μg/ml blasticidin S (Wako Pure Chemical Industries, Ltd, Osaka, Japan). The blasticidin-resistant cells were pooled and analyzed.
To establish stable A549 cells, we used a lentiviral vector (the pLVX series, TaKaRa) to express small hairpin RNAs (shRNAs) under the control of the U6 promoter (the target sequences knocked down are listed in Table
5
Overexpression of lncRNA LINC00501
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The specific short hairpin RNA (shRNA) directed against human lncRNA LINC00501 was cloned into pENTR TM/U6 plasmid (GenePharma, Shanghai, China) and called sh-LINC00501, and non-targeted shRNA (sh-NC, GenePharma) was adopted as negative control.MiR-129-5p mimics (miR-129-5p-mimics), inhibitors (miR-129-5p-inhibit), and their respective controls (miR-NC) were all processed by RiboBio (Guangzhou, China). For overexpression of HMGB1, the full-length HMGB1 sequence was transfected into pcDNA vector (ThermoFisher, China), called pcDNA-LINC00501, and pcDNA-3.1 was adopted as blank control. Transfection operations were all carried out with Lipofectamine 3000 reagent (Life Technologies Corp., Carlsbad, United State) under the manufacturer’s instruction. Stable transfected H1299 and A549 cells were collected, and cultured in medium containing 0.5 mg/mL G418 (Sigma-Aldrich, st Louis, Missouri, Untied States), and stable transfected cells were adopted for later analyses.
6
Overexpression of NEAT1 and BCL2 in Ovarian Cancer
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To overexpress NEAT1, NEAT1 genomic fragment was cloned by polymerase chain reaction (PCR) and inserted into the pCMV6 empty vector (Vector; OriGene, Rockville, MD, USA), named as pCMV6-NEAT1 (NEAT1). pcDNA-BCL2 plasmid was established by cloning BCL2 sequence into the empty pcDNA Vector (Thermo Fisher Scientific). siRNA against NEAT1 (si-NEAT1), scrambled siRNA negative control (si-NC), miR-34a-5p mimic (miR-34a-5p), scrambled miRNA negative control (miR-NC), miR-34a-5p inhibitor (anti-miR-34a-5p), and anti-miRNA negative control (anti-miR-NC) were obtained from Genepharma (Shanghai, China). SKOV3 and OVCAR3 cells were transfected with oligonucleotide or plasmids using Lipofectamine 2000 (Thermo Fisher Scientific).
7
Modulating NFAT5 Pathway via miR-142-5p
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MiR-142-5p inhibitor and mimic (anti-miR-142-5p and miR-142-5p), as well as their matching NCs (anti-miR-NC and miR-NC), were synthesized by Sangon (Shanghai). To generate the pcDNA-NFAT5 (NFAT5) plasmid, the full-length sequence of NFAT5 was inserting into the pcDNA vector (Thermo Fisher). Transient transfection was conducted using the Lipofectamine 3000 reagent (Thermo Fisher).
8
Overexpression and Silencing of PPA1 in Ovarian Cancer Cell Lines
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IOSE-386 (OSE), a normal ovarian epithelial cell line, was obtained from the China Center for Type Culture Collection (CCTCC, Shanghai, China); serous ovarian carcinoma (SOC) cell lines (A2780 and OVCAR3) were purchased from American Type Culture Collection (ATCC, USA). All cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) with10% fetal bovine serum (FBS) at 37°C, 5% CO 2 , with 100 IU/mL penicillin and 100 μg/mL streptomycin.
Human PPA1 cDNA was isolated using RT-PCR and subcloned into pcDNA vector (#V79020, Thermo Fisher Scientific, PA, USA) to generate the pcDNA-PPA1 construct, which was later used for overexpression transfection with empty vector pcDNA as control. Small interfering RNA (siRNA) for PPA1 (#105886) and negative control siRNA (ncRNA) were also ordered from Thermo Fisher Scientific. Briefly, A2780 and OVCAR3 cells were seeded into six-well plates, and then the cells were transfected with each plasmid or siRNA by Lipofectamine 3000 (Invitrogen, PA, USA) following the manufacturer's instructions. After transfection, cells were incubated for another 48 h and subjected to Western blot analyses.
Human PPA1 cDNA was isolated using RT-PCR and subcloned into pcDNA vector (#V79020, Thermo Fisher Scientific, PA, USA) to generate the pcDNA-PPA1 construct, which was later used for overexpression transfection with empty vector pcDNA as control. Small interfering RNA (siRNA) for PPA1 (#105886) and negative control siRNA (ncRNA) were also ordered from Thermo Fisher Scientific. Briefly, A2780 and OVCAR3 cells were seeded into six-well plates, and then the cells were transfected with each plasmid or siRNA by Lipofectamine 3000 (Invitrogen, PA, USA) following the manufacturer's instructions. After transfection, cells were incubated for another 48 h and subjected to Western blot analyses.
9
miR-518a-3p and TMEM2 Regulation in Breast Cancer
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MiR-518a-3p mimics, inhibitor and corresponding negative controls (NC mimic, NC inhibitor), as well as shRNAs targeting TMEM2 (shTMEM2 #1 or #2), were synthesized by GenePharma (Suzhou, China). Full length TMEM2 was constructed and cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). MDA-MB-453 or MDA-MB-468 cells were transfected with miR-518a-3p mimics, inhibitor or corresponding negative controls, shRNAs, or pcDNA-TMEM2 and pcDNA vector via Lipofectamine 3000 (Invitrogen). For the treatment with JAK1 inhibitor, MDA-MB-468 cells were cultured in RPMI 1640 medium containing 100 nM Pyridone 6 (Sigma-Aldrich, St. Louis, MO, USA), and then transfected with miR-518a-3p mimics, NC mimic, pcDNA-TMEM2 or pcDNA vector.
10
Overexpression of miR-532-5p and CCND1 in Cells
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Cells (1×105 cells/well) were seeded into 6-well plates and cultured for 24 h at 37°C with 5% CO2. Cell transfection was performed when the cells reached 80% confluency. The miR-532-5p mimic and mimic-negative control (mimic-NC; Invitrogen; Thermo Fisher Scientific, Inc.) were transfected directly into cells. For overexpression of miR-532-5p, the cells were transfected with mimic at a final concentration of 25 nM for 48 h at 37°C. For pcDNA-NC (empty vector, Invitrogen; Thermo Fisher Scientific, Inc.) and pcDNA-CCND1, full length transcript of CCND1 was amplified from cDNA obtained from 293T cells by PCR using PrimeSTAR® HS DNA polymerase (Takara Bio, Inc.), and was transfected at a final concentration of 500 ng for 48 h at 37°C. The PCR amplification product was inserted into the KpnI and BamHI sites of the pcDNA vector (Invitrogen; Thermo Fisher Scientific, Inc.), which was termed pcDNA-CCND1. Transfection was performed using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Transfection efficiency was determined by RT-qPCR 48 h after transfection.
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