The immortalized HAC cell line (CHON-001; CRL-2846; ATCC, Manassas, VA, USA) was cultured in DMEM (M22650; R&D systems) containing 0.1 mg/ml G418 disulfate salt (4131; R&D systems) and 10% FBS (R&D systems). HAC cells were used to transiently transfect with exogenous nucleotides or vectors using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for 36 h. The oligonucleotides were circ_0045714-siRNA (si-circ_0045714), PIK3R3-siRNA (si-PIK3R3), negative control (NC)-siRNA (si-NC), miR-331-3p mimic, miR-NC mimic, miR-331-3p inhibitor (anti-miR-331-3p), and miR-NC inhibitor (anti-miR-NC). The vectors were empty pCD5-ciR vector (GENESEED, Guangzhou, China), recombinant pCD5-ciR-circ_0045714 vector, pmiR-Reporter vector (Promega, Madison, WI, USA) expressing circ_0045714 containing the wild type (WT) or mutant type (MUT) of miR-331-3p response elements, and pmiR-Reporter vector (Promega, Madison, WI, USA) expressing PIK3R3 3’UTR containing WT or MUT of miR-331-3p response elements. Single and co-transfection models were performed per the instructions, and transfected cells at 36 h were harvested for further assays.
Pmir reporter vector
Manufactured by Promega
Sourced in United States
The PMIR-reporter vector is a plasmid designed for the expression and detection of miRNA (microRNA) activity. It contains a promoter that drives the expression of a reporter gene, which is repressed by the insertion of a miRNA target sequence. This allows for the monitoring of miRNA expression or activity in cells transfected with the vector.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (5 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Renilla luciferase reporter vector, by Promega (1 mentions)
Fetal bovine serum (fbs), by R&D Systems (1 mentions)
M22650, by R&D Systems (1 mentions)
Chon 001, by American Type Culture Collection (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Fluorescence multi detection microplate reader, by Agilent Technologies (1 mentions)
Dual luciferase assay, by Promega (1 mentions)
13 protocols using pmir reporter vector
1
Regulation of circRNA-miRNA-mRNA Axis in HAC Cells
2
Luciferase Assay for miRNA Regulation
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Briefly, DNA oligonucleotides and pMiR-Reporter vectors (Promega, Madison, WI, USA) were used to construct luciferase reporter vectors (pMiR-HAGLROS-wt/pMiR-HAGLROS-mut and pMiR-SMARCA5-wt/pMiR-SMARCA5-mut). pMiR-HAGLROS-wt or pMiR-HAGLROS-mut and miR-100 mimetics or negative controls (NC) were co-transfected into HEK293 cells. The luciferase activity was determined 48 h after the transfection using Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA).
3
miR-622 Binding Site Assay
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The circ-0006091 or CCNB1 3’ UTR fragments with the WT or MUT miR-622 binding sites were placed in the pMIR reporter vectors (Promega, Madison, WI, USA). Subsequently, the HCCLM3 and Huh-7 cells were transfected with the vectors mentioned above and the miR-622 mimic or miR-NC. After 48 h, a Dual-Luciferase Reporter Assay System (Promega) was used to assay luciferase activity.
4
miR-30c Regulates ASF/SF2 Gene
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Using the databases TargetScan (http://targetscan.org/ ), PicTar (http://pictar.mdc-berlin.de/ ) and miRDB (http://www.mirdb.org/miRDB/ ), the putative target genes of miR-30c were investigated, and the ASF/SF2 gene was identified among them. To amplify the 3′UTR sequence of the human ASF/SF2 gene, primers were designed. Mlu1 and Spe1 sequences were cloned into the ends of primers (Promega Corporation, Madison, WI, USA). According to the manufacturer's protocol, 3′-UTR cloning was performed in a pMIR reporter vector (Promega Corporation). The pMIR-ASF/SF2 3′-UTR reporter vector (100 ng) was cotransfected with 50 ng of β-Gal and miRNA-30c mimic or anti-miR-30c using Lipofectamine 2000 transfection reagent. All Stars negative control siRNA (Qiagen, Inc.) and miScript inhibitor negative control (Qiagen, Inc.) were used as controls for mimic or inhibitor transfection, respectively. At 24 h post-transfection, luciferase reporter assays were performed, as described previously (9 (link)).
5
Validating miR-365 Binding to HOXA9 3'-UTR
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Validation of miRNA targets was performed by cloning partial HOXA9 3′-UTRs containing the sequence recognized by the miR-365 core seed region. The oligos carrying native and mutated miR-365 binding sites with SpeI and SacI restriction sites for cloning were annealed and cloned into the pMIR-reporter vector (Promega). The oligos used in this study are listed in (Supplementary Table 2 ).
For dual luciferase reporter assays, HEK293T cells were cotransfected with pMIR-reporter constructs, the Renilla luciferase reporter vector (Promega), and either 150 nM miR-365 or control mimic (Guangzhou Ribobio), using Lipofectamine 2000 reagent (Life Technologies), according to the manufacturer’s instructions. Luciferase activity was measured at 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s protocol. Correction for differences in transfection efficiency was performed by normalizing firefly luciferase activity to total Renilla luciferase.
For dual luciferase reporter assays, HEK293T cells were cotransfected with pMIR-reporter constructs, the Renilla luciferase reporter vector (Promega), and either 150 nM miR-365 or control mimic (Guangzhou Ribobio), using Lipofectamine 2000 reagent (Life Technologies), according to the manufacturer’s instructions. Luciferase activity was measured at 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s protocol. Correction for differences in transfection efficiency was performed by normalizing firefly luciferase activity to total Renilla luciferase.
6
Validating hsa_circ_0091994-miR-324-5p-HMGA1 Interaction
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The potential downstream miRNA target of hsa_circ_0091994 was predicted using Starbase (http://starbase.sysu.edu.cn/ ) and CircInteractome (https://circinteractome.nia.nih.gov/index.html ) online databases. The potential downstream target of miRNA was predicted with TargetScan (http://www.targetscan.org/vert_72/ ), miRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/micrornapredictedtarget.html ), and miRDB (http://www.mirdb.org/ ). Luciferase reporter assay was performed to verify the potential targeting relationship mentioned above. All oligos were synthesized by GenePharm (Shanghai, China). The oligos containing wild type or mutant 3’ UTR binding site of hsa_circ_0091994 were cloned into pMIR-reporter vector (Promega, Madison, WI, USA). Then, pMIR-reporter vector constructs, renilla luciferase reporter vector, and miR-324-5p agomir were co-transfected into AGS cells for 48 hr. Dual Luciferase Reporter Assay System (Promega, Madison) was used once the relationship between miR-324-5p and HMGA1 was verified. The results were presented as a ratio of firefly luciferase activity normalized to Renilla luciferase activity [23 (link)].
7
Sirt1 3' UTR Luciferase Assay
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The 3′ UTR fragment of Sirt1 was amplified using specific primers and cloned into the multiple cloning site of pMir-reporter vector (Promega, Madison, WI, USA). Mutations in the 3′ UTR fragment were generated via PCR-based mutagenesis. The primers used are shown in Supplementary information, Table S1 . The AML12 cells were co-transfected with the plasmids and Gly-tRF mimics or inhibitors. After 48 h, the cells were collected and luciferase assays were performed according to the manufacturer’s protocol (Promega).
8
Validation of Notch1 as miR-744 Target
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TargetScan (www.targetscan.org/ ) and miRanda (www.microrna.org ) were applied to predict the potential targets of miR-744. Notch1 was predicted as a potential target of miR-744 and was selected for experimental confirmation. The 3′-UTR of human Notch1 containing the wild type or mutant predicted that binding sequences of miR-744 was cloned by GenePharma, inserted into the p-MIR-reporter vector (Promega, Manheim, Germany), and named as p-MIR-WT-Notch1-3′-UTR and p-MIR-MUT-Notch1-3′-UTR, respectively. For luciferase reporter assay, cells were plated into 24-well plates and then cotransfected with miR-744 mimic or miR-NC and p-MIR-WT-Notch1-3′-UTR or p-MIR-MUT-Notch1-3′-UTR using Lipofectamine 2000 following the manufacturer’s instructions. Luciferase activity was determined at 48 h posttransfection using the Dual-Luciferase Reporter Assay System (Promega) in accordance with the manufacturer’s protocol. Renilla luciferase activity was normalized to firefly luciferase activity.
9
Validating miRNA-mRNA interactions
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NBAT-1 and miR-4504 potential targets were predicted with the miRDB MicroRNA Target Prediction Database (http://mirdb.org/ ), respectively. For the dual-luciferase reporter assay, NBAT-1 fragments or the 3′-UTR of WWC3 that carried miR-4504 binding were inserted into the pMIR-reporter vector (Promega, Madison, Wisconsin, USA). OXA-resistant CRC cells were cotransfected with pMIR-wild-type (WT)/mutant (Mut) vector (100 ng) with miR-4504 mimics or miR-NC (50 nM) using the Lipofectamine 2000. After transfection for 48 h, Renilla and Firefly luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega).
10
Luciferase Assay for miRNA Binding
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The oligos containing the native or mutant binding site were cloned into pMIR-reporter vector (Promega). HEK293T cells were seeded into 12 well plates and co-transfected with pMIR-reporter constructs, renilla luciferase reporter vector, miR-27a mimic or NC mimic. Luciferase activities were measured at 48 h after transfection. The firely luciferase activity was normalized to renilla luciferase activity. The sequences of those oligos are listed in Supplementary Material .
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