The luciferase reporter assay was performed as previously described [38 (link)]. Briefly, A549 and H1299 lung cancer cells were seeded into 24-well tissue culture plates to get a 40–60% confluence 24 h later. Cells were transfected with pBIIx-luc NF-κB-dependent reporter construct and the Renilla luciferase vector (Promega, Madison, WI, USA). At 24 h post-transfection, the cells were treated with vehicle (DMSO, 0.1% v/v concentration), HKLM (2 × 108 cells/mL), and poly(I:C) (25 µg/mL) in the presence or absence of different concentrations of propionate for 24 h. The cells were lysed, and luciferase activity was measured using a dual luciferase assay kit (Promega). Ctrl A549, FFAR2KO A549, Ctrl H1299, and FFAR2KO H1299 cells were transfected with pBIIx-luc NF-κB-dependent reporter construct and the Renilla luciferase vector (Promega). At 24 h post-transfection, the cells were treated with vehicle (DMSO, 0.1% v/v concentration), HKLM (108 cells/mL), and poly(I:C) (20 µg/mL) in the presence or absence of propionate (1 mM) for 6 h. The cells were lysed, and luciferase activity was measured using a dual luciferase assay kit (Promega). The luciferase assay was carried out in triplicate in at least three independent experiments.
Dual luciferase assay kit
Manufactured by Promega
Sourced in United States, Germany, China, United Kingdom, Japan, France
The Dual Luciferase Assay Kit is a laboratory tool that measures the activities of two different luciferase reporter enzymes simultaneously within the same sample. The kit provides reagents and protocols to quantify the activities of firefly and Renilla luciferase reporters, allowing for normalization of experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (262 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (110 mentions)
Passive lysis buffer (plb), by Promega (100 mentions)
Pgl3 basic vector, by Promega (58 mentions)
Prl tk, by Promega (48 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (41 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (31 mentions)
Psicheck 2, by Promega (26 mentions)
Glomax 20 20 luminometer, by Promega (26 mentions)
Prl sv40, by Promega (21 mentions)
Pgl3 vector, by Promega (21 mentions)
Lipofectamine, by Thermo Fisher Scientific (21 mentions)
Pmirglo vector, by Promega (21 mentions)
Luminoskan ascent microplate luminometer, by Thermo Fisher Scientific (20 mentions)
Psicheck 2 vector, by Promega (20 mentions)
Glomax 96 microplate luminometer, by Promega (19 mentions)
Pgl3 luciferase reporter vector, by Promega (17 mentions)
Pmirglo, by Promega (16 mentions)
Opti mem, by Thermo Fisher Scientific (15 mentions)
Dual luciferase reporter assay system, by Promega (15 mentions)
1 920 protocols using dual luciferase assay kit
1
NF-κB Activation Assay for Lung Cancer
2
Identifying STAT3 Target Genes: Sox4 and NLRP3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The downstream target genes for transcription factor STAT3 were identified by JASPAR 2022 (https://jaspar.genereg.net/ ). We first constructed the wild type (WT) and mutant (Mut) Sox4 promoter plasmids (pGL4.1-Sox4-promoter-WT or pGL4.1-Sox4-promoter-MUT) by referring to the binding sites between STAT3 and Sox4 promoter region. 293 T cells were then co-transfected with the corresponding plasmids and internal plasmids (pTK-RL) containing the Renilla luciferase gene by applying Lipofectamine 3000 (Invitrogen). After 48 h of transfection, luciferase (Firefly/Renilla) activity was measured using the Dual luciferase assay kit (Promega).
For NLRP3, the wild type promoter of NLRP3 was constructed (pGL4.1-NLRP3-promoter-WT). SCC-15 and SCC-25 cells were then co-transfected with pGL4.1-NLRP3-promoter-WT plasmids, different concentration of Sox4 overexpression plasmids (0, 0.1, 0.5, 1, 5, 10, 20, 50 ng) and internal plasmids (pTK-RL) containing the Renilla luciferase gene by applying Lipofectamine 3000 (Invitrogen). After 48 h of transfection, luciferase (Firefly/Renilla) activity was measured using the Dual luciferase assay kit (Promega).
For NLRP3, the wild type promoter of NLRP3 was constructed (pGL4.1-NLRP3-promoter-WT). SCC-15 and SCC-25 cells were then co-transfected with pGL4.1-NLRP3-promoter-WT plasmids, different concentration of Sox4 overexpression plasmids (0, 0.1, 0.5, 1, 5, 10, 20, 50 ng) and internal plasmids (pTK-RL) containing the Renilla luciferase gene by applying Lipofectamine 3000 (Invitrogen). After 48 h of transfection, luciferase (Firefly/Renilla) activity was measured using the Dual luciferase assay kit (Promega).
3
HNRNPA3 3'UTR Luciferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Luciferase reporter vectors containing HNRNPA3 3′-UTR-WT or 3′-UTR-Mut were co-transfected with control mimic or miR-218-5p mimic in HEK293T cells. At 24 h post-transfection, the cell lysate supernatant was analyzed using the dual luciferase assay kit (Promega). The values of luciferase obtained from the assays were normalized with Renilla luciferase activity.
To detect the activation of the SREBF1 promoter, cells were transfected with the luciferase reporter vectors, phRL-TK plasmids, and HNRNPA3-siRNA or NC-siRNA. Then, cells were untreated or treated with PEDV for 24 h. Cell lysates were prepared and analyzed by a dual luciferase assay kit (Promega).
To detect the activation of the SREBF1 promoter, cells were transfected with the luciferase reporter vectors, phRL-TK plasmids, and HNRNPA3-siRNA or NC-siRNA. Then, cells were untreated or treated with PEDV for 24 h. Cell lysates were prepared and analyzed by a dual luciferase assay kit (Promega).
4
Validating miR-3591-5p Regulation by m6A Modification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To further validate the effect of m6A modification on miR-3591-5p expression, m6A modification sites on pri-miR-3591 sequences were predicted through SRAMP. We identified one putative m6A recognition sites on pri-miR-3591 sequences. According to this predicted results, mutagenesis from adenosine to thymine was generated by QuikChange II Site-Directed Mutagenesis Kit (Agilent, USA) in the light of the instruction, and then the wild-type and mutant pri-miR-3591 reporter vectors were co-transfected with shFTO plasmid into chondrocytes. Transfer after 48 h, luciferase activities were assessed by a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) in accordance with the instructions. For the verification of miR-3591-5p and PRKAA2 targeted interactions, the wild-type or mutant-type of PRKAA2 (containing the binding site of miR-3591-5p) was cloned into the luciferase vector, and then transfected into in chondrocytes together with miR-3591-5p mimics or NC mimics, respectively. Transfer after 48 h, luciferase activities were assessed by a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) on the base of the instructions. Finally, the signal value of Renilla luciferase was normalized as an internal reference.
5
Luciferase Assay for miR-219-5p Targeting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect the prediction between miR-219-5p and LRH-1-3′-UTR, a cDNA fragment of the LRH-1-3′-UTR mRNA containing the seed sequence of the mature miR-219-5p binding site or a mutated binding site of the 3′-UTR sequence was cloned into the pmirGLO dual-luciferase vector (Promega, Madison, WI, USA). The constructed pmirGLO dual-luciferase vector was cotransfected with miR-219-5p mimics into 293T cells using Lipofectamine 2000 (Invitrogen). After incubation for 48 h, cells were harvested, and the relative luciferase activity was measured using the dual-luciferase assay kit (Promega). To detect the Wnt signaling activity, cells were cotransfected with phRL-TK Renilla luciferase vectors (Promega), TOPFlash firefly luciferase reporter vector (Addgene, Cambridge, MA, USA), and miR-219-5p mimics or anti-miR-219-5p. After 48 h, the relative luciferase activity was determined using the dual-luciferase assay kit (Promega).
6
Validating miR-200b-3p Targeting of TBK1 via Luciferase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Luciferase reporter vectors containing WT TBK1 3′ UTR or MUT TBK1 3′ UTR were co-transfected with the control mimic or miR-200b-3p mimic into HEK-293T cells to validate the miR-200b-3p targeting TBK1. The transfected cell lysates were analyzed by using the dual luciferase assay kit (Promega) at 24 h post-transfection. All obtained luciferase values were normalized against the Renilla luciferase control.
HEK-293T cells grown in 48-well plates were co-transfected with luciferase reporter plasmids (IFN β-Luc, IRF3-Luc, ISRE-Luc, or NF-κB-Luc) and the pRL-TK plasmid to detect activation of the IFN pathway, along with the indicated amount of empty vector or miRNAs. 293T cells were left untreated or were treated with SeV for additional 12 h. The dual luciferase assay kit (Promega) was used to prepare and analyze cell lysates for firefly and Renilla luciferase activities.
HEK-293T cells grown in 48-well plates were co-transfected with luciferase reporter plasmids (IFN β-Luc, IRF3-Luc, ISRE-Luc, or NF-κB-Luc) and the pRL-TK plasmid to detect activation of the IFN pathway, along with the indicated amount of empty vector or miRNAs. 293T cells were left untreated or were treated with SeV for additional 12 h. The dual luciferase assay kit (Promega) was used to prepare and analyze cell lysates for firefly and Renilla luciferase activities.
7
Dual Luciferase Assay for Transfection Efficiency
8
Validating miRNA-CNTN1 Interaction and IFN-I Pathway Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate the miRNA targeting CNTN1, luciferase reporter vectors containing WT CNTN1 3’-UTR or the mutant were co-transfected with the control mimic or miR-200c mimic into HEK293 cells. At twenty-four hours after transfection, the transfected cell lysates were analyzed by using the dual luciferase assay kit (Promega). All obtained luciferase values were normalized against those of the Renilla luciferase control.
To detect activation of the IFN-I pathway, HEK293 cells grown in 24-well plates were co-transfected with Luciferase reporter plasmids (IFN-β-Luc, ISRE-Luc, or STAT1-Luc) and the pRL-TK plasmid, along with the indicated amount of empty vector or plasmids expressing CNTN1 or other molecules. At 24 h post-transfection, the cells were left untreated or were treated with SeV, IFN-β, or IAV for an additional 12 h. Cell lysates were prepared and analyzed for firefly and Renilla luciferase activities by using the dual luciferase assay kit (Promega).
To detect activation of the IFN-I pathway, HEK293 cells grown in 24-well plates were co-transfected with Luciferase reporter plasmids (IFN-β-Luc, ISRE-Luc, or STAT1-Luc) and the pRL-TK plasmid, along with the indicated amount of empty vector or plasmids expressing CNTN1 or other molecules. At 24 h post-transfection, the cells were left untreated or were treated with SeV, IFN-β, or IAV for an additional 12 h. Cell lysates were prepared and analyzed for firefly and Renilla luciferase activities by using the dual luciferase assay kit (Promega).
9
3'UTR Luciferase Assay for miRNA Targeting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids containing either wild-type or mutated 3′-UTR sequence were transfected into HEK293T cells using Lipofectamine™ 2000 Transfection Reagent (Invitrogen) and Opti-MEM (Invitrogen) as instruction, and renilla plasmid was co-transfected as the transfection control. The cells were also co-transfected with either the negative control or the mimic (10 pmol/ml) and incubated for 24 h, and then lysed using 1 × Passive Lysis Buffer (Promega Dual Luciferase Assay Kit, Promega). The lysis was applied to luciferase activity test using a luminometer (Orion II Luminometer) according to the manufacturer’s instructions. Firefly luciferase values were normalized to those of Renilla luciferase.
10
Transient Transfection of HNF4α and PGC-1α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells were transiently transfected with pHNF4‐tk‐luc (Yoshida et al. 1997 ), pcDNA‐Flag‐HNF4α, and pcDNA‐NT‐PGC‐1α‐HA. pRL‐SV40 control plasmid expressing Renilla luciferase was used for normalization. The firefly luciferase activity was determined 48 h after transfection using a Promega Dual‐Luciferase assay kit (Promega, Madison, WI) and normalized using Renilla luciferase activity. Data represent mean ± SEM of at least three independent experiments.
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!