The part of VDR 3′-UTR containing the putative binding site and the 5′ and 3′ flanking sequence was amplified by a pair of primers (F: 5′-GAC GTT CTA GAT GAG TCA TGA TCT CCC TGC C-3′, R: 5′-GAC GTT CTA GAT ACC CTA CAT CAC GGA ACC C-3′) and subcloned into pGL3-control vector (Promega, USA) immediately downstream of the luciferase gene to form the pGL3-VDR-3′-UTR construct. Point mutations in seed sequence were generated by PCR to form the pGL3-VDR-3′-UTR-MUT construct. 1 × 105 cells were plated in 24-well plates for 24 h. 0.4 μg of pGL3 constructs plus 0.07 μg of pRL-CMV plasmid-expressing renilla luciferase (Promega) were transfected in combination with 60 pmol of either a negative control RNA oligonucleotide or miR-675 mimics/inhibitors (GenePharma, Shanghai, China) using Lipofectamine 3000 (Invitrogen). After 48 h, luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega, USA). Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well. The results were obtained from three independent experiments and each one was performed in triplicate.
Prl cmv plasmid expressing renilla luciferase
Manufactured by Promega
Sourced in China, United States
The PRL-CMV plasmid-expressing renilla luciferase is a laboratory tool that expresses the renilla luciferase reporter gene under the control of the cytomegalovirus (CMV) promoter. Renilla luciferase is a bioluminescent protein that catalyzes the oxidation of coelenterazine, resulting in the emission of light. This plasmid can be used for various applications, such as monitoring gene expression, reporter assays, and live-cell imaging.
Automatically generated - may contain errors
2 protocols using prl cmv plasmid expressing renilla luciferase
1
Dual-Luciferase Assay for miR-675 Targeting VDR 3'UTR
2
Validating miR-497 Regulation of HDGF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A search with TargetScan (http://www.targetscan. org) predicted HDGF as a direct target gene of miR-497. A seven-nucleotide seed match of position 37-43 of the HDGF 3'-UTR with miR-497 was revealed. In order to verify this interaction, a luciferase reporter assay was performed. Cells were plated in a 12-well plate at ~90% confluence and transfected with miR-497 mimics or NC using Lipofectamine 2000 (Invitrogen). For the reporter assays, stably miR-497-overexpressing cells were transiently transfected with reporter plasmids driven by a wild-type or mutated fragment from the 3'UTR of HDGF. The luciferase reporter vectors, including HDGF-3'UTR Wt and HDGF-'UTR Mut, were obtained from GenePharma Co., Ltd. (Shanghai, China). Each sample was also co-transfected with 0.05 µg pRL-CMV plasmid expressing Renilla Luciferase (Promega Corp., Madison, WI, USA) as an internal control for transfection efficiency. After 48 h of incubation, the cells were harvested and lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corp.) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Each assay was replicated three times.
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!