TargetScan software (version 7.0; targetscan.org/ ) (18 (link)) was used to identify the miR-15b-5p binding site in the 3'-UTR of ACSS2. The wild-type (WT) and mutant (MUT) 3'-UTR sequences of ACSS2 at the miR-15b binding site were chemically synthesized by General Biosystems, Inc., and inserted into the XhoI/NotI site of psiCHECK-2 luciferase vector (Promega Corporation) to construct the recombinant plasmids psiCHECK2-ACSS2-WT and psiCHECK2-ACSS2-MUT, respectively. miR-15b-5p mimics or miR-15b-5p mimic NC were co-transfected with psiCHECK2-ACSS2-WT and psiCHECK2-ACSS2-MUT into HEK-293T cells using the Exfect Transfection reagent (Vazyme Biotech Co., Ltd.). After 48 h, firefly and Renilla luciferase activities were measured on an EnSpire reader (PerkinElmer, Inc.) using the Dual Luciferase Assay System (Dalian Meilun Biology Technology Co., Ltd.).
Protocol detail
Validating miR-15b-5p Regulation of ACSS2
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psiCHECK-2 luciferase vector
Exfect Transfection reagent
EnSpire reader
Dual Luciferase Assay System
293t cells
Acss2
Assay
Binding site
Firefly
Luciferase
Plasmids
Promega
Renilla luciferase
Transfection
Vector
Corresponding Organization :
Other organizations : Shanghai First People's Hospital, Shanghai Jiao Tong University, Pudong Medical Center, Fudan University
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Variable analysis
independent variables
- MiR-15b-5p mimics
- MiR-15b-5p mimic NC
- Firefly luciferase activity
- Renilla luciferase activity
- PsiCHECK2-ACSS2-WT
- PsiCHECK2-ACSS2-MUT
- None specified
- MiR-15b-5p mimic NC
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