The UCSC website (http://genome.ucsc.edu/ ) was used to predict the binding sites between ESR1 and lncRNA MEG3. The sequences of wild-type (WT) and mutated (MUT) lncRNA MEG3 promoter were synthesized and inserted into pGL3 vectors for constructing the corresponding reporters. Subsequently, these recombinant reporters were cotransfected with pcDNA3.1/ESR1 or pcDNA3.1 into HepG2 cells. After 48 h of transfection, a dual-luciferase detection kit was used to evaluate the luciferase activity using a dual luciferase reporter analysis system (Promega). Renilla luciferase expression vector pRL-TK (Takara, Beijing, China) was used as an internal control.
Dual luciferase detection kit
Manufactured by Promega
Sourced in United States
The Dual Luciferase Detection Kit is a laboratory instrument designed to measure the activity of two different luciferase reporter enzymes simultaneously within the same biological sample. The kit provides reagents and protocols to quantify the activity of firefly and Renilla luciferase reporters, which are commonly used to study gene expression and cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Pmirglo, by Promega (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Pmirglo luciferase vector, by Promega (4 mentions)
Prl tk, by Takara Bio (3 mentions)
Lipofectamine 3000 transfection kit, by Promega (3 mentions)
Pmirglo vector, by Promega (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Glomax 20 20 luminometer, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pcrii topo 2 luciferase receptor vectors, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter analysis system, by Promega (1 mentions)
Psicheck, by Thermo Fisher Scientific (1 mentions)
Pmir report vector, by Promega (1 mentions)
Ripa lysate, by Thermo Fisher Scientific (1 mentions)
Primary antibodies against mtor, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Mtt kit, by Merck Group (1 mentions)
Ht 29, by Shanghai Institute of Biochemistry and Cell Biology (1 mentions)
Sw620, by Shanghai Institute of Biochemistry and Cell Biology (1 mentions)
Transwell insert, by Merck Group (1 mentions)
49 protocols using dual luciferase detection kit
1
Predicting ESR1-lncRNA MEG3 Binding Sites
2
Dual-Luciferase Reporter Assay for linc01833 and S100A4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this study, the dual-luciferase detection kit (Promega, Madison, USA) was used for relevant experimental studies and to analyze the dual-luciferase reporter activity. Specifically, A549 and HCC4006 cells were co-transfected with linc01833 WT, linc01833 mut, miR-519e-3p, S100A4 WT, S100A4 mut, or NC siRNA. Afterwards, cells were digested, dissolved, and centrifuged to collect supernatant, followed by measurement of renilla and firefly luciferase activity by microplate photometer (BioTeck, Winooski, VT, USA). All the experiments were performed in triplicate, followed by calculation of the average value to ensure the reliability of the experimental results.
3
Smad2 Regulatory Relationship with miR-152-3p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The targeted regulatory relationship between Smad2 and miR-152-3p was further determined by constructing the reporter gene plasmid of the target gene, Smad2. Specifically, the wild-type (WT) fragment of Smad2 3′ UTR was amplified using the primers containing the miR-152-3p and Smad2 binding site (Table 2 ). Then, the Smad2 3′ UTR mutant-type (MUT) fragment was synthesized by mutating 4 bases of the binding site using the Stratagene mutation kit (Stratagene, Heidelberg, Germany). Subsequently, these two fragments were connected to the luciferase reporter vector pmiRGLO (Promega, Madison, WI, USA) and the recombinant plasmids pmiRGLO–Smad2-3′ UTR-WT and pmiRGLO–Smad2-3′ UTR-MUT were constructed. The extracted recombinant plasmid was then co-transfected with the miR-152-3p mimic into the HEK-293T cells. After 48 h of transfection, the activity of luciferase was detected using a dual-luciferase detection kit (Promega, Madison, WI, USA).
4
miRNA-197-3p Binding Mechanism Study
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MALAT1 Wt was obtained by amplifying the MALAT1 fragments that included binding sites with miR-197-3p via PCR, and MALAT1 Mut was constructed similarly other than mutating the binding sites. Then cells at the logarithmic phase were classified under treatments of pmirGLO-MALAT1 Wt+miR-197-3p mimic, pmirGLO-MALAT1 Wt+miR-NC, pmirGLO-MALAT1 Mut+miR-197-3p mimic and pmirGLO-MALAT1 Mut+miR-NC. In addition, p120-ctn Wt was gained by amplifying the p120-ctn fragments that included binding sites with miR-197-3p via PCR. The p120-ctn Mut was constructed analogous, except that the binding sites were mutated. This series of cells were categorized into pmirGLO-p120-ctn Wt+miR-197-3p mimic group, pmirGLO-p120-ctn Wt+miR-NC group, pmirGLO-p120-ctn Mut+miR-197-3p mimic group and pmirGLO-p120-ctn Mut+miR-NC group. After 24-h transfection, we measured the fluorescence intensity of each group in accordance with the procedures of dual luciferase detection kit (Promega, USA).
5
Regulation of Foxo3 by miR-29c-3p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The prediction and analysis of miR-29 c-3p with Foxo3ʹs targeting binding sites were via Bioinformatics website RNA22, and the cloning of corresponding sequences was into luciferase reporter vector pmirGLO (Promega Corp., Madison, WI, USA). The co-transfection of Foxo3-Wide type (WT)/mutant (MUT), miR-29 c-3p mimic/NC was into KGN cells. And then the evaluation of the relative luciferase activity was via a dual luciferase detection kit (Promega Corp., Madison, WI, USA).
6
Luciferase Assay for circCSPP1, ROCK1, and ZEB1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The luciferase assay was performed using the dual-luciferase reporting system psiCHECK (Thermo Fisher Scientific, Inc.). The wild-type (WT) or mutant-type (mut) sequences of circCSPP1, ROCK1 and ZEB1 were cloned into the psiCHECK2 plasmid. 293 T cells (ATCC, 2 × 104 cells/well) were cultured overnight in 24-well plates. The cells were transfected with the WT or mut reporter vector along with miR-431 mimics (10 nM) or mimics control (10 nM) using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.). Finally, the luciferase activity of cells was detected with a Dual-Luciferase Detection kit (Promega Corporation) after 48 h of transfection. The data were quantified by normalizing to Renilla luciferase activity.
7
Dual-Luciferase Reporter Assay for Transcriptional Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human cells were seeded at a density of 6 × 104 per 0.5 ml in phenol red-free DMEM medium containing 5% charcoal-treated FBS in 24-well cell culture plates. The cells were co-transfected with ERα, GR or PR mammalian expression plasmids (only for HEK293T cells), as well as both a luciferase reporter plasmid and a constitutive Renilla luciferase expression plasmid (pRL-CMV). In another experiment, cells were transfected with luciferase reporter plasmids as described for Supplementary Fig. 9d along with pRL-CMV. At 48 h post-transfection, cells were lysed with Passive Lysis Buffer, and firefly and Renilla luciferase activities were measured using the Dual-Luciferase detection kit (Promega) with a bioluminescence plate reader. Renilla luciferase activity was used as transfection control.
8
Molecular Analysis of miR-296-3p Regulation on TGIF1 3'UTR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR amplification was utilized to generate the 3′-untranslated region (UTR) of TGIF1 containing the binding sites for miR-296-3p. The mutant type (MUT) TGIF1 3′-UTR was constructed by the QuikChange Site-Directed Mutagenesis Kit from Stratagene in the light of manufacturer’s instructions. The obtained TGIF1 3′-UTR sequence and the MUT TGIF1 3′-UTR sequence were inserted into a pmiR-REPORT vector (Promega Corporation, Madison, WI, USA). Then, the WT TGIF1 3′-UTR plasmid and MUT TGIF1 3′-UTR plasmid were transfected with miR-296-3p mimic and its NC into the SW480 cells and HCT-116 cells in compliance with the instructions of Lipofectamine™ 2000 transfection reagent. After 48 h, the samples were collected and the relative luciferase activities of firefly and renilla fluorescence were measured in compliance to the instructions of the dual luciferase detection kit (Promega). Each reaction was run in triplicate to obtain the average value.
9
Investigating Colon Cancer Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Healthy colonic epithelial cells (HCoEpiC), human colon cancer cell lines HT‐29, SW620, and SW480, and LoVo and Coco‐2 cell lines were purchased from Shanghai Institute of Cell Biology. Fetal bovine serum (FBS) and DMEM/F12 were purchased from Hyclone; Cytiva. TRIzol®, Lipofectamine® 2000, RIPA lysate, and BCA protein assay kit were purchased from Invitrogen; Thermo Fisher Scientific, Inc. Pcmv plasmid, miR‐96‐5p mimics/inhibitor, and irrelevant nucleic acid sequence were purchased from Shanghai GenePharma Co., Ltd., and Annexin‐V/PI Apoptosis kit was purchased from CapitalBio Technology, Inc. Matrigel was purchased from BD Biosciences, the MTT kit was purchased from Sigma‐Aldrich; Merck KGaA, the Transwell inserts was purchased from MilliporeSigma and the dual‐luciferase detection kit was purchased from Promega Corporation. Primary antibodies against mTOR, LC 3, LC 3B, Beclin1, P62, and GAPDH were purchased from Abcam. mTOR agonist was purchased from BIOLEBO.
10
Evaluating lncRNA-miRNA Interaction Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The target sequence was predicted by the bioinformatics website, and lncRNA SDCBP2-AS1 or EPDR1 3′-UTR sequence binding to miR-100-5p was amplified and mutated via site-directed mutagenesis kit (NBS, Beijing) to generate lncRNA SDCBP2-AS1 or EPDR1 3′-UTR mutant (MUT) sequence. Subsequently, the amplified lncRNA SDCBP2-AS1 or EPDR1 3′-UTR sequence and the lncRNA SDCBP2-AS1 or EPDR1 3̲′-UTR MUT sequence were inserted into the psi-CHECK2 reporter (Promega, WI, USA) and sequenced. Wild-type (Wt)-lncRNA SDCBP2-AS1 or Wt-EPDR1 3′-UTR and the Mut-lncRNA SDCBP2-AS1 or Mut-EPDR1 3′-UTR vectors were collected. Wt-lncRNA SDCBP2-AS1 or Wt-EPDR1 3′-UTR vector, or Mut-lncRNA SDCBP2-AS1 or Mut-EPDR1 3′-UTR vector, and miR-100-5p mimic or mimic-NC were co-transfected into cells via LipofectamineTM 2000. The samples were harvested 48 h after transfection. Finally, the relative luciferase activity of firefly and Renilla was detected with the dual luciferase detection kit (Promega).
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!