The cloned CTSD gene containing 200 bp of the coordinated lysosomal expression and regulation (CLEAR)-box sequence (gccacgtgag, the specific sequence region of TFEB binding) was inserted into the luciferase vector pGL3-basic (Promega, Madison, WI) and named CLEAR-box+Empty. The CLEAR-box sequence was mutated (TAACTAGTTA) and named CLEAR-box-mut+Empty. The recombinant TFEB sequence was inserted into pcDNA3.1 and named CLEAR-box+TFEB and CLEAR-box-mut+TFEB. A mixture of plasmids (plasmid ratio CLEAR-Box:TFEB:TK = 5:5:1, total plasmid amount: 2 μg) and 2 μL of Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) were added to the medium of 293 cells for cotransfection. Then, 15, 30, or 60 μM 3,4-DC was added 24 h after transfection, and the samples were named CLEAR-box+TFEB+15 μM, CLEAR-box+TFEB+30 μM, and CLEAR-box+TFEB+60 μM, respectively. The cells were collected 48 h after transfection, and a luciferase detection kit (Beyotime, RG027) was used to detect firefly luciferase and Renilla luciferase intensities. The relative luciferase intensities were calculated.
Luciferase detection kit
Manufactured by Beyotime
Sourced in China, United States
The Luciferase detection kit is a laboratory tool designed to quantify the presence and activity of luciferase, a bioluminescent protein commonly used as a reporter in biological and biochemical assays. The kit provides the necessary reagents and protocols to detect and measure luciferase levels in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Pmir reporter, by Huayueyang (3 mentions)
Pgl3 basic, by Promega (2 mentions)
Glomax 20 20 luminometer, by Promega (2 mentions)
Glomax 20 20 fluorescence detector, by Promega (2 mentions)
Psicheck 2 vector, by Promega (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Pmir rb report, by RiboBio (1 mentions)
Pmir rb report vector, by RiboBio (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pgl3 promoter vector, by GenScript (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Glomax20 20 luminometer fluorescence detector, by Promega (1 mentions)
Pmir rb report plasmid, by RiboBio (1 mentions)
17 protocols using luciferase detection kit
1
CLEAR-box Regulates TFEB Activity
2
Luciferase Reporter Assay for miRNA Targeting
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The synthetic 3′UTR fragment of genes was inserted into the pMIR-reporter (Huayueyang Biotechnology Co., Ltd., Beijing China) using two endonuclease sites Spe I and Hind III. The mutant (Mut) site of complementary sequence of the seed sequence was designed based on the IL-12A-wild type (Wt). After restrictive endonuclease digestion, the target fragment was inserted into the pMIR-reported using T4 DNA ligase. The recombinant luciferase reporter plasmids IL-12A-Wt and IL-12A-Mut with the correct sequences were respectively co-transfected with miR-21 into human embryonic kidney 293T cells (CRL-1415, Shanghai Xin Yu Biological Technology Co., Ltd., Shanghai, China). After 48 h of transfection, the PMNs were lysed, and the luciferase activity was measured using a Glomax20/20 luminometer (Promega Corp., Madison, WI, USA) based on the instructions of the luciferase detection kit (RG005, Beyotime Biotechnology Co., Shanghai, China). The interaction between XIST and miR-21 was evaluated using the same method. The experiment in each group was repeated three times.
3
Luciferase Assay for miR-124 and STAT3 3'UTR
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The sequence of wild type (WT) of miR-124 and STAT3 mRNA 3′-untranslated region (3′-UTR) and sequence of mutant type (MUT) after site-directed mutation of WT target site were synthesized. pmiR-RB-REPORT™ plasmid (RiboBio Co., Ltd., Guangzhou, China) was digested by restriction endonuclease. Then the synthetic target gene fragments WT and MUT were inserted into pmiR-RB-REPORT™ vector (RiboBio Co., Ltd., Guangzhou, China). At the same time, the empty plasmid was transfected as the control group, and the correct luciferase reporter plasmids WT and MUT were used for subsequent transfection. The vectors of MUT and WT were co-transferred to 293 T cells with mimic-NC or miR-124 mimic together with oe-NC or oe-ZFAS1, respectively. The cells were collected and lysed after 48-h transfection, and the supernatant was obtained by centrifugation for 3–5 min. Luciferase detection kit (RG005, Beyotime Biotechnology Co., Ltd., Shanghai, China) was applied for determining the relative lights units (RLU). Using firefly luciferase as an internal parameter, the RLU value determined by renilla luciferase was divided by the RLU value measured by firefly luciferase, and the relative fluorescence value was obtained.
4
VvWRKY5 Transcriptional Regulation Assay
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The VvWRKY5 CDS was inserted into the pRI101 vector as an effector. The VvJAZ2 and VvMYC2 promoter fragments were inserted into the pGreenII0800-LUC vector as reporters. The helper plasmid pSoup was used to transform the recombinant vectors into the A. tumefaciens GV3101 before being injected into N. benthamiana leaves. Luciferase signaling was observed using a live fluorescence imager (Tanon-5200, Shanghai, China), whilst LUC/REN activity was also measured using a luciferase detection kit (Beyotime, Shanghai, China). Supplementary Data Table S3 shows the details of all primers used here.
5
Transient Transcriptional Regulation Assay
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The VvUFGT and VvLOX promoter fragments (~2000 bp upstream of ATG) were fused to the pGreenII0800-LUC vectors to produce the reporters (proVvUFGT-LUC and proVvLOX-LUC). The VvMYBA1 and VvWRKY5 CDSs were fused to the pRI101-AN vectors to produce the effectors (35S::VvMYBA1 and 35S::VvWRKY5). These recombinant constructs were introduced into Agrobacterium GV3101. Next, a mixed Agrobacterium suspension (OD600 = 1) harboring the recombinant plasmids was transiently injected into N. benthamiana leaves. These tobacco plants were then grown in dark conditions. Two days after infection, luciferase signaling was detected by a Tanon-5200 imaging device (Shanghai, China), while promoter activity based on the LUC/REN ratio was assessed employing a luciferase detection kit (Beyotime, Shanghai, China). Relevant primers utilized are provided in Table S1 (see online supplementary material).
6
Investigating PINK1 3'-UTR Regulation by miR-421
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The target site wild-type (WT) sequence of the PINK1 3′-untranslated region (3′-UTR) and its site-directed mutation sequence (MUT, sequence: 5′-CCGTCA-3′) were synthesized. The restriction endonuclease was adopted to digest the pGL3-BASIC plasmid (Guangzhou Ribio Biotechnology Co., Ltd., Guangzhou, China), and then the synthesized target gene fragments WT and MUT were inserted into the pmiR-RB-REPORTTM vector (Ribio). Next, the empty plasmid was transfected as the control group, and the correctly sequenced luciferase reporter plasmid was used for subsequent transfection. The vectors were separately transferred with miR-421 mimic to NSCs. After 48 h of transfection, the cells were collected, lysed, and centrifuged for 5 min. Then, the relative light unit (RLU) value of the supernatant was examined by the luciferase detection kit (RG005, Beyotime Biotechnology, Shanghai, China). The degree of activation of the target reporter gene was compared, based on the ratio of the RLU value of β-cal luciferase activity to the RLU value of firefly luciferase activity.
7
Luciferase Assay for miR-598 Binding
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Human THBS2 3′-UTR sequence or the mutant (MUT) sequence of THBS2 3′-UTR, containing the predicted binding sites of miRNA-598, was inserted into the pGL3 promoter vector (Genscript, Nanjing, China). The 293 T cells were seeded in 24-well plates (5 × 105 cells/well) on the day before transfection. Subsequently, the cells were co-transfected with luciferase reporter vectors (0.12 µg) and miR-598 mimic or NC using the Lipofectamine 3000 Reagent (Invitrogen, USA). Luciferase reporter assay was conducted 48 h after transfection according to the manufacturer’s instructions. The Luciferase detection kit (RG005, Beyotime) was used to detect the luciferase activity in a Glomax20/20 fluorescence detector (Promega).
8
Nrf2 Gene Regulation by miR-142-5p
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Wild (WT) and mutant (MUT) sequences of the Nrf2 gene were designed according to the binding site of miR-142-5p in the 3′-UTR of Nrf2 with reference to Targetscan 7.2. The Vectors of Nrf2-WT and -MUT were conducted by GenePharma using pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, United states). 293 T cells were transfected with the modified vectors and miR-142-5p mimics/mimics-NC using Lipofectamine 3000 (Invitrogen, United states). Following the instruction of luciferase detection kit (Beyotime, China), the luciferase activity in each group was then detected in TriStar2 S microplate reader (Berthold Technology, Germany). The ratio of Firefly/Rennila activity was calculated. Dual-Luciferase reporter gene assay was performed in triplicates.
9
Sp5 3'UTR Luciferase Assay
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The artificially synthesized Sp5 3′‐untranslated region (UTR) fragments were introduced into the psiCHECK‐2 vector (Promega Corporation, Madison, WI, USA). The complementary sequence mutation site was designed on the Sp5 wild type (WT), which was inserted into psiCHECK‐2 vector reporter plasmid. The correctly sequenced luciferase reporter plasmid Sp5 3′UTR‐WT (100 ng) and Sp5 3′UTR‐mutant type (MUT) (100 ng) were transfected with miR‐486‐5p mimic and mimic NC (2 nmol/L; Dharmacon Research, Inc, Waltham, MA, USA) into HEK‐293T cell (CRL‐1415; Shanghai Xin Yu Biotech Co., Ltd, Shanghai, China). After 48 hours, cells were collected and lysed. The luciferase activity was detected using a luciferase detection kit (RG005; Beyotime Biotechnology, Shanghai, China) on a Glomax20/20 fluorescence detector (Promega Corporation).
10
Dual-Luciferase Assay for miR-576-5p Binding to ANXA2 3'UTR
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The sequences of wild-type (WT) and mutant (MUT) ANXA2 3ʹUTR comprising miR-576-5p binding sites were synthesized and inserted into pMIR-REPORTTM Luciferase vector plasmid (Ambion, Austin, TX, USA) to establish ANXA2-WT and ANXA2-MUT. ANXA2-WT or ANXA2-MUT reporter plasmid, along with an miR-576-5p mimic or mimic NC was co-transfected into 293 T cells through Lipofectamine 2000 (Invitrogen).Cells were lysed after 48 h of transfection and centrifuged for 3–5 min to take the supernatant. Renilla luciferase buffer and firefly luciferase buffer were dissolved using the luciferase detection kit (Beyotime). The buffer (100 μL) was added with a substrate at 1:100 to prepare Renilla luciferase detection working solution. The sample (20–100 μL) from each group was added with 100 μL of the firefly luciferase detection reagent and detected by using Luminometer TD-20/20 Fluorometer (Promega, USA). The cell lysate was used a blank control. A Luciferase detection working solution (100 μL) was added, and a relative light unit (RLU) was measured. Renilla luciferase (Renilla) expression vector PRL-TK was considered as the internal reference. The RLU value obtained after the measurement was divided by the RLU value obtained after the measurement with Renilla luciferase to obtain the luciferase activity.
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