The 3ʹuntranslated region fragment of IL1R1 was predicted, and amplified by PCR. Co-transfection of firefly luciferase reporters (LINC01123-wide-type [WT], LINC01123-mutant-type [MUT], IL1R1-WT, IL1R1-MUT), renilla luciferase vector (pMIR-Report Luciferase, Promega) and mimic NC or miR-125a-3p mimic was performed in HEK293T cells according to the instruction of Lipofectamine 2000 (Invitrogen, USA). firefly and renilla luciferase activities were analyzed on a Spectra ™ single tube multimode reader (Sunnyvale, CA, USA).
Pmir report luciferase
Manufactured by Promega
Sourced in United States
The PMIR-Report Luciferase is a lab equipment product designed for bioluminescence-based assays. It functions as a reporter system that measures gene expression levels by detecting and quantifying luciferase activity.
Automatically generated - may contain errors
4 protocols using pmir report luciferase
1
Luciferase Assay for miRNA-mRNA Interaction
2
Luciferase reporter assay for miR-223-3p targets
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For luciferase reporter assays, the 3’UTR segments of FOXO1 predicted to interact with miR-223-3p were amplified by PCR. Cells were cotransfected with firefly luciferase reporter plasmid (lncRNA GUSBP5-AS-WT, lncRNA GUSBP5-AS-MU, FOXO1-WT, FOXO1-MU) and a Renilla luciferase vector (pMIR-Report Luciferase, Promega) plus negative control and small RNAs (NC, miR-223-3p mimics) using Lipofectamine 2000 (Invitrogen, USA). Experiments were performed at least three times. Cells were harvested 48 h later, and firefly and Renilla luciferase activities were analyzed with a Modulus™ single tube multimode reader (Sunnyvale, CA, USA).
3
Luciferase Reporter Assay for PTEN
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For the luciferase reporter assay, cells were cotransfected with firefly luciferase reporter plasmid (PTEN‐WT, PTEN‐WUT) and a Renilla luciferase vector (pMIR‐Report Luciferase, Promega) plus negative control and small RNAs (NC, miR‐205 mimics) using HiTrans™ LipoPlus Reagent (SYNTHGENE). Firefly and Renilla luciferase activities were analysed with Multi‐Detection Readera (SpectraMax5). Experiments were performed at least three times.
4
Validation of miR-124-3p Target Genes
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GRB2 and AKT3 as direct target genes of miR-124-3p were screened out based on the TargetScan database. The luciferase reporter gene assay was performed with the Dual-Luciferase Reporter Assay System (Promega). The full length of the 3′-untranslated region (UTR) of the wild-type (WT) GRB2/AKT3 gene was cloned into the vector pMIR-REPORT™ Luciferase (Promega), and the mutant-type (MUT) vectors were constructed by site-directed mutation of the binding site between miR-124-3p and GRB2/AKT3. The phRL-TK vector (Promega) expressing the Renilla luciferase was used to normalize the transfection efficiency. An equivalent of 1 × 104 BV2 microglia were seeded in 96-well plates. At 70–80%, the reporter gene vectors (WT and MUT of GRB2/AKT3) were co-transfected with miR-124-3p-mimic/mimic-NC into the BV2 microglia to determine the luciferase activity, according to the manufacturer’s instructions.
+ Expand
GRB2 and AKT3 as direct target genes of miR-124-3p were screened out based on the TargetScan database. The luciferase reporter gene assay was performed with the Dual-Luciferase Reporter Assay System (Promega). The full length of the 3′-untranslated region (UTR) of the wild-type (WT) GRB2/AKT3 gene was cloned into the vector pMIR-REPORT™ Luciferase (Promega), and the mutant-type (MUT) vectors were constructed by site-directed mutation of the binding site between miR-124-3p and GRB2/AKT3. The phRL-TK vector (Promega) expressing the Renilla luciferase was used to normalize the transfection efficiency. An equivalent of 1 × 104 BV2 microglia were seeded in 96-well plates. At 70–80%, the reporter gene vectors (WT and MUT of GRB2/AKT3) were co-transfected with miR-124-3p-mimic/mimic-NC into the BV2 microglia to determine the luciferase activity, according to the manufacturer’s instructions.
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