To assess miR‐223 repressing the 3′ UTR of FoxO1 mRNA in Jurkat cells, 1.5 × 106 cells were transfected with 200 ng of pMIR‐REPORT™ Reporter Vector (Ambion Inc.) or 200 ng of pMIR‐REPORT‐FoxO1‐3′ UTR, 10 ng of Renilla luciferase reporter pSV40‐RL (transfection control; Promega) and 50 nM of miR‐223‐3p precursor molecules or equal amounts of miRNA precursor molecules‐negative controls. At 48 hrs after transfection, luciferase assays were performed using the Dual‐Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. All experiments were carried out five times.
Pmir report reporter vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PMIR‐REPORT™ Reporter Vector is a plasmid-based tool designed for gene expression studies. It contains a reporter gene that can be used to measure and quantify gene expression levels in experimental systems. The core function of this product is to serve as a reporter for monitoring transcriptional activity, without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Psv40 rl, by Promega (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Glomax, by Promega (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Prl tk vector, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
5 protocols using pmir report reporter vector
1
MiR-223 Regulation of FoxO1 3'UTR in Jurkat Cells
2
Evaluating miR-765 Regulation of BRD4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pMIR-REPORT Reporter vector (Ambion; Thermo Fisher Scientific, Shanghai, China) containing BRD4’s 3’-UTR sequence with the predicted miR-765 binding sites, or pMIR-BRD4-3ʹ-UTR, was provided by Dr. Zhao [28 (link)]. The construct was transfected to OS cells by Lipofectamine 3000. Cells were then infected with lv-pre-miR-765 or lv-antagomiR-765 for 48h. Thereafter, cells were harvested and assayed for the measurement of luciferase activity using a Dual-Luciferase Reporter Assay System (Promega, Shanghai, China).
3
Luciferase Assay to Validate miRNA-mRNA Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The segment of the 3'-UTR of RANKL predicted to interact with miR-338-3p or a mutated sequence containing the potential binding region were synthesized by PCR and inserted via the BglΙΙ and HindΙΙΙ restriction sites downstream of the luciferase open reading frame into the pMiR-Report reporter vector (Ambion Inc., Austin, TX, USA). The correct clones were confirmed by sequencing. Pre-OCs were seeded onto a 24-well plate (1 x 10 4 cells per well), and were co-transfected with a luciferase reporter construct (200 ng) and miRNA mimic/inhibitor (50 nM) using Lipofectamine 2000 according to manufacturer instructions. Luciferase activity was measured 2 days after transfection using the Dual-Luciferase Assay System (Promega, Madison, WI, USA). Luminescent signals were quantified by a luminometer (Glomax, Promega), and each value from the firefly luciferase construct was normalized to those of Renilla luciferase in the assay.
4
Evaluating circRNA-miRNA-mRNA Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to reveal the circRNA-miRNA interaction, has-circRNA_0046367 (circBase, Rajewsky lab, Berlin, Germany) sequence containing the putative target sites for miR-34a was synthesized and cloned into the pMIR-REPORT™ reporter vector (Thermo Fisher Scientific Inc., Waltham, USA) downstream to the firefly luciferase (pMIR-REPORT-circRNA_0046367-wildtype). Mutant version of circRNA_0046367 (pMIR-REPORT-circRNA_0046367-mutant) was also generated with the deletion of complementary sites. After the cotransfection of reporter vector (pMIR-REPORT-circRNA_0046367-wildtype or pMIR-REPORT-circRNA_0046367 -mutant) and oligonucleotides (miR-34a mimics or negative control) in 293T cells, firefly luciferase activity was subjected to measurement by dual-luciferase assay kit (Promega, Madison, USA) against that of renilla luciferase [30 (link)]. Similarly, the complementarity between miR-34a and 3′-untranslated region (3′-UTR) of PPARα was evaluated by the methods mentioned above. Each assay was repeated in 5 independent experiments.
5
Interaction Between circPhc3 and miR-93-3p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To reveal the interaction between circPhc3 and miR-93-3p, a circPhc3 sequence containing the complementary sequences of miR-93-3p was chemically synthesized and cloned downstream of the firefly luciferase gene in the pMIR-REPORT™ reporter vector (Thermo Fisher Scientific, Inc.). After incubation in DMEM (Thermo Fisher Scientific, Inc.) supplemented with 0.5% FBS for 48 h, 293T cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences; 1×105 cells/well) were co-transfected with 1 µg pMIR-circPhc3, 50 ng pRL-TK vector (Promega Corporation) and 100 nM miR-93-3p mimic or miR-NC using Lipofectamine 3000 for 48 h at 37°C. At 48 h post-transfection, cells were collected and luciferase activities were measured using a dual luciferase reporter assay kit (Promega Corporation). Firefly luciferase activities were normalized to Renilla luciferase activities.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!