To detect the interaction between RB1CC1 and miR-141-5p, the full-length 1847 bp 3′UTR of wild-RB1CC1 (WT) and same length mutant-RB1CC1 (MUT) were amplified and then cloned into pmiR-Report Luciferase vector (GenScript, Nanjing, China). The PSCs were co-transfected 50 nM miR-141-5P mimic or 100 nM miR-141-5p inhibitor with 500 ng of Luciferase constructs according to the manufacturer’s protocol. The cells were harvested 24 h after transfection, and the luciferase activity was measured with a Dual-Luciferase Reporter Assay System (Promega, Madison, USA), through Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher Scientific, Massachusetts, USA). Firefly luciferase activity was used to normalize the transfection efficiency. The sequences were listed in Supplementary Table S2 .
Pmir report luciferase vector
Manufactured by GenScript
Sourced in China
The PmiR-Report Luciferase vector is a plasmid-based reporter system designed for the study of microRNA (miRNA) activity. It contains a luciferase reporter gene that can be used to quantify the effects of miRNAs on gene expression. The vector allows for the insertion of miRNA target sequences, enabling the user to assess the impact of specific miRNAs on the expression of the luciferase reporter.
Automatically generated - may contain errors
2 protocols using pmir report luciferase vector
1
Detecting miR-141-5p Interaction with RB1CC1
2
Investigating miR-30a-5p Binding Interactions
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Full‐length MUT was amplified and cloned into the pmiR‐Report luciferase vector (GenScript). According to the predicted binding site of TPT1‐AS1 and miR‐30a‐5p, the sequence of the binding site was mutated to CACCCGT. Then, 50 nm miR‐30a‐5p mimic and 500 ng of luciferase constructs were cotransfected into PANC‐1 cells, and 100 nm miR‐30a‐5p inhibitor and 500 ng of luciferase constructs were cotransfected into BxPC‐3 cells according to the manufacturer's instructions. The luciferase activities were measured with the Dual‐Luciferase Reporter Assay System (Promega) after 24 hours transfection using a Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher Scientific) according to the manufacturer's instructions. Firefly luciferase activity was used to normalize the transfection efficiency. For examining the binding between ITGB3 and miR‐30a‐5p, ITGB3‐WT or ITGB3‐MUT luciferase reporter was built to perform similar procedures. The plasmids that inserted four fragments of the promoter (P1, P2, P3, and P4) in front of the luciferase sequence were built to explore which fragments of the promoter could bind to STAT3.
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