Luciferase reporter plasmid was constructed by cloning human VDAC1 mRNA 3′-UTR into pMIR-Report construct (Ambion, Austin, USA). Wild type or mutant VDAC1 mRNA fragment was amplified and cloned into the luciferase reporter via SpeI and HindIII sites. All the primers were listed in Table 2 . Luciferase reporter assays were performed using Dual-Luciferase Reporter Assay System (Promega). Briefly, HEK 293T cells plated in a 96-well plate were co-transfected with 50 nM miR-320a mimics or negative control oligonucleotides, 20 ng of firefly luciferase reporter and 10 ng of pRL-TK (Promega, USA) using the INTERFERin reagent (Polyplus-transfection, France). Cells were collected 24 hours after transfection for luciferase assay.
Pmir report construct
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PMIR-Report construct is a laboratory equipment designed for the analysis and quantification of protein-metabolite interactions. It employs a proprietary technique to capture and measure these interactions, providing researchers with valuable insights into the complex relationships between proteins and metabolites within biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (7 mentions)
Prl tk, by Promega (5 mentions)
Jetprime reagent, by Polyplus Transfection (4 mentions)
Interferin reagent, by Polyplus Transfection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Jetpei, by Polyplus Transfection (1 mentions)
Luciferase reporter assay system, by Promega (1 mentions)
Prl sv40, by Promega (1 mentions)
10 protocols using pmir report construct
1
Luciferase Assay for VDAC1 mRNA 3'UTR
2
Luciferase Reporter Assay for miR-1271 Targeting
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The 3′UTR fragments of CCNG1 containing putative binding sites for miR-1271 were cloned into pMIR-Report construct (Ambion, Austin, TX). The primers (Biomart, Shanghai, China) were constructed according to previous reports [33 (link),34 (link)]. Mutant 3′UTR of CCNG1, which carried a mutated sequence in the complementary site for the seed region of miR-1271, were generated using the fusion PCR method. Luciferase reporter assay was performed in HEK293 cells as described previously [12 (link)].
3
Characterizing miR-429 Binding to ZEB1 3'UTR
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The 3′UTR fragments of ZEB1 containing putative binding sites for miR-429 were cloned into pMIR-Report construct (Ambion, Austin, TX). The primers were constructed by Biomart (Shanghai, China) according to previous publications [38 (link),39 (link)] and the details are provided in previous papers [39 (link),40 (link)]. Mutant 3′UTR of ZEB1, which carried a mutated sequence in the complementary site for the seed region of miR-429, was generated using the fusion PCR method. Luciferase reporter assay was performed in HEK293 cells as described previously [41 (link)].
4
Luciferase Reporter Assay for miR-542-3p Targeting Smad2
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The pMIR-Smad2 luciferase reporter vector was constructed by cloning a human Smad2 mRNA sequence into a pMIR-Report construct (Ambion; Thermo Fisher Scientific, Inc.). A 60-bp Smad2 mRNA fragment (5,084–5,143 bp of Smad2 mRNA), which is the predicted target of miR-542-3p, was amplified and cloned into the luciferase reporter via SpeI and HindIII sites. All sequences are listed in Table I . Luciferase reporter assays were performed as follows: 5×103 293T cells were seeded in a 96-well plate, then co-transfected with 50 nM single-stranded miR-542-3p mimics or negative control oligonucleotides, 10 ng of pMIR-Smad2 luciferase reporter, pMIR luciferase reporter and 3 ng of pRL-TK (Promega Corporation, Madison, WI, USA) using the JetPRIME® reagent (Polyplus-transfection SA). Cells were collected 36 h after transfection and analyzed using a Dual-Luciferase Reporter assay system (Promega Corporation). pRL-TK was cotransfected as an internal control to correct the differences in both transfection and harvest efficiencies. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity.
5
Luciferase Reporter Assay for miRNA Targeting
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The luciferase reporter construct was developed by cloning the human ADM 3′-UTR sequence into the pMIR-Report construct (Ambion, United States) via SacI and HindIII sites. All primers are listed in Supplementary Table 10 . Briefly, HEK-293 cells were plated in a 48-well plate and co-transfected with 50 nM miRNA mimics or negative control oligonucleotides (Sangon, China), 200 ng of firefly luciferase reporter and 50 ng of pRL-TK (Promega, United States) using the JetPRIME reagent (Polyplus, United States). Cells were collected 36 h after the last transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega, United States).
6
Investigating VDAC1 3'UTR Regulation
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Both wild-type (Wt) and mutant (Mut) VDAC1 3'- untranslated regions (UTRs) were amplified by PCR and cloned into the pMIR-Report construct (Ambion, USA). For luciferase assay, HCT116 and SW480 cells plated in 24 well plates were co-transfected with Wt or Mut VDAC1 plasmid along with miR-mimic or NC respectively. Transfection was conducted using Lipofectamine 2000 in accordance with the procedure of manufacture (Invitrogen, USA). Cells were collected at 48h after transfection for luciferase reporter assay analyzed using a Dual-luciferase Reporter Assay system (Promega, USA), and renilla luciferase was used for normalization.
7
Luciferase Assay for BRAF mRNA
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Wild-type (WT) or mutant (Mut) BRAF mRNA fragment was amplified and cloned into pMIR-Report construct (Ambion, Austin, TX, USA). Primers used for the BRAF WT mRNA fragment were: 5′-CCC AAG CTT AGG ACC TCA GCG AGA AAG GAA GTC AT-3′ (sense) and 5′-CTA GAC TAG TAC ATC ACC ATG CCA CTT TCC CTT G-3′ (antisense). Primers used for the BRAF Mut mRNA fragment were: 5′-CCC AAG CTT ACC TGG ACA GCC TCA AAG GAA GTC AT-3′ (sense) and 5′-CTA GAC TAG TAC ATC ACC ATG CCA CTT TCC CTT G-3′ (antisense). The vectors were verified by direct sequencing. HEK 293 T cells were placed onto 24-well plate and co-transfected with pMIR-BRAF-WT mRNA reporter plasmids (100 ng) or pMIR-BRAF-Mut mRNA reporter plasmids (100 ng), pMIR-TK (25 ng) and miR-378-5p mimics or negative control oligonucleotides (50 nM) using jetPEI (Polyplus). After 24 hours, cells were harvested and the reporter activity was detected using Dual-luciferase reporter assay system (Promega, Madison, Wisconsin, USA).
8
Luciferase reporter assay for TIAM1 mRNA
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By cloning human TIAM1 mRNA sequence into pMIR-Report construct (Ambion, Austin, USA), luciferase reporter construct was made. Wild type or mutant TIAM1 mRNA fragment (from 26 to 32) was amplified and cloned into the luciferase reporter via SpeI and HindIII sites. Using the JetPRIME reagent (Polyplus-transfection), HGC-27 cells were co-transfected with 20 nM single-stranded miRNA mimics or negative control oligonucleotides, 20 ng of firefly luciferase reporter and 10 ng of pRL-TK (Promega, USA). Cells were collected after being transfected for 24 hours and analyzed using Dual-Luciferase Reporter Assay System (Promega).
9
Luciferase Assay for BCL2 3'-UTR
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A luciferase reporter plasmid was established through cloning the 3′-UTR of BCL2 or the mutated sequence into the pMIR-Report construct (Ambion; Thermo Fisher Scientific, Inc.). The miR-371-5p mimics, or the corresponding control were transfected together with a reporter plasmid and pRL-SV40 (Promega Corporation, Madison, WI, USA) into 5-8F cells. After 48 h, the luciferase activity was measured using the Luciferase Reporter Assay system (Promega Corporation) in accordance with the manufacturer's protocol.
10
Luciferase Assay for miRNA-3'UTR Interaction
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We prepared the 3′-UTR luciferase reporter construct by cloning the human mRNA 3′-UTR sequence into the pMIR-Report construct (Ambion, Austin, TX, USA). The Atg7 3′-UTR fragment (from 1236 to 1643) was amplified and cloned into the luciferase reporter via SacII and EcoRI sites. Next, oligonucleotides bearing the designed sequence were inserted at the same site to obtain the Atg7 3′-UTR point mutation. Luciferase reporter assays were performed as reported previously (Wu et al. 2013) (link). Briefly, MMQ and GH3 cells plated in a 24-well plate were cotransfected with 100 nM single-stranded miRNA mimics, 25-ng firefly luciferase reporter comprising Atg7 3′-UTR and 6-ng pRL-TK (Promega) using the JetPRIME reagent (Polyplus-transfection). After 36-h transfection, cells were collected and analyzed using the Dual-Luciferase Reporter Assay System (Promega).
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