The wild type and mutant 3’UTR of BRCA1 were constructed into psi-check-2 vector (Promega, Madison, WI). Cells were plated in 24-well plates and the plasmids (psi-check-2-WT or psi-check-2-mut) were transfected using lipofectamine® 3000 (#11668019, ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Luciferase activity was measured by Dual-Luciferase Assay reagent (Promega) according to the manufacturer’s manual. The wild type and mutant promoter of miR-146a-5p were constructed into pGL3 vector (Promega). The pGL3-WT or pGL3-mut were co-transfected with pRL-TK using lipofectamine® 3000. Luciferase activity was measured by Dual-Luciferase Assay reagent (Promega) according to the manufacturer’s manual.
Dual luciferase assay reagent
Manufactured by Promega
Sourced in United States, China
The Dual-Luciferase® Reporter Assay System is a laboratory tool that allows for the quantitative measurement of firefly and Renilla luciferase activities within the same sample. It provides a method for normalizing experimental variations and enables the rapid quantification of gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Passive lysis buffer (plb), by Promega (6 mentions)
Glomax 20 20 luminometer, by Promega (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Victor x2 2030, by PerkinElmer (4 mentions)
Lipofectamine, by Thermo Fisher Scientific (4 mentions)
Infinite m200, by Tecan (3 mentions)
Polarstar omega, by BMG Labtech (3 mentions)
Multimode plate reader, by PerkinElmer (2 mentions)
Modulus luminometer, by Promega (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Pwpi luc vector, by Promega (2 mentions)
Pgl3 basic vector, by Promega (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Prl sv40 encoding renilla luciferase rluc, by Promega (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Fluorescence flx800 microplate fluorescence reader, by Agilent Technologies (1 mentions)
Phrg tk renilla, by Promega (1 mentions)
Glomax 96 microplate luminometer, by Promega (1 mentions)
91 protocols using dual luciferase assay reagent
1
Dual-Luciferase Assay for BRCA1 3'UTR and miR-146a-5p promoter
2
Cloning and Validating miR-101 Regulation of CDK8
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The human wild CDK8 3′ untranslated region (UTR) (base 92–99, 5′-GUACUGUA-3′) was amplified and cloned into the multiple cloning sites in a psi-CHECKTM-2 luciferase miRNA expression reporter vector (Promega). Site-directed mutagenesis of the miR-101 target site in the CDK8-3′-UTR (5′-AUGCGGCA-3′) was used as a negative control and termed CDK8-3′UTR mutant. The primers selected were as follows: CDK8-3′UTR, 5′-ATGCACTGTTGCGAATGCTG-3′ (forward) and 5′-AATGCTTGCCCCTAGCACAT-3′ (reverse); CDK8-3′UTR mutant, 5′-GAGAATATGCGGCAACAACC-3′ (forward) and 5′-GGTTGTTGCCGCATATTCTC-3′ (reverse). For the reporter assays, cells were transiently transfected in 24-well plates with luciferase reporter gene constructs and has-miR-101, or an antagomir that was designed to target endogenous has-miR-101, using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activities for each transfected well were measured 48 h after transfection using dual luciferase assay reagents (Promega). Three independent transfection experiments were performed in triplicate for each plasmid construct.
3
FAS Promoter Luciferase Reporter Assay
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The human FAS promoter reporter construct (FAS-luc) was kindly given by Prof Qiang Liu (University of Saskatchewan, Canada)60 (link). MIHA cells were co-transfected FAS-luc and pRL-SV40 encoding Renilla luciferase (rLuc) (Promega). Tandem repeat of ARE was inserted into the pGL6 luciferase reporter vector (ARE-pGL6-luc) (Beyotime). MIHA cells were co-transfected the ARE-pGL6-luc and pRL-SV40 encoding Renilla luciferase (rLuc) (Promega) before receiving FFA treatment. Control cells were co-transfected with empty pGL3-basic luciferase reporter vector and pRL-SV40. Dual-Luciferase assay was performed with Dual-Luciferase assay reagents (Promega). The luciferase readings for each sample were normalized against the rLuc levels. All samples were analyzed in triplicate in each individual experiment.
4
Transient Transfection of CV-1 Cells
5
Transcriptional Regulation of Stress Genes
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Transcription activity of SiNF-YA1 or SiNF-YB8 against promoters of stress responsive marker genes was performed using dual luciferase assay of transiently transformed tobacco leaves (Huang et al., 2013 (link)). Marker gene promoters from tobacco were amplified by thermal asymmetric inter-laced PCR (Tail-PCR) and subcloned into the transient expression reporter vector pGreenII0800-LUC which contained the CaMV 35S promoter-REN cassette and the promoterless-LUC cassette. pBI121-SiNF-Y vector was used as effector construct as previously described (Yotsui et al., 2013 (link)). No-effector construct was used as control. Firefly luciferase and renilla luciferase were assayed using the dual luciferase assay reagents (Promega, USA). Data was collected as the ratio of LUC/REN. Three independent experiments were accomplished and for each sample three technical replicates were analyzed.
6
Transient Expression Assay for SlPIN1 Promoter
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The full-length SlARFs ORF were amplified and cloned into the effector vector, pGreen II 62-SK. A –1479 bp fragment (upstream from the start codon) from the SlPIN1 promoter was amplified and cloned into the reporter vector, pGreen II 0800-LUC. Both the effector and reporter vector were respectively co-transformed into the Agrobacterium tumefactions strain GV3101 cells with the pSoup vector, then infiltrated into N. benthamiana young leaves and incubated 72 h in the dark. LUC and REN were analyzed using the dual luciferase assay reagents (Promega) with an Infinite M200 (Tecan). All primers used in this analysis are listed in Supplementary Table S2 .
7
Transcriptional Regulation of Stilbene Synthase
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The promoter sequences containing the W-box (TTGACC or TTGACT) of VvSTS9 and VvSTS48 were inserted into the pGreen II 0800-LUC vector to serve as reporters, and the CDS of VqWRKY31 was inserted into the pGreen II 62-SK vector to serve as the effector. Specific primers used in these constructions are listed in Supplementary Table S1 . These plasmids were transformed into A. tumefaciens GV3101 along with the helper plasmid pSoup-p19 (#AC1003, Weidi, Shanghai, China). Four-week-old tobacco leaves were co-infiltrated with Agrobacterium harboring the effector plasmid and different reporter plasmids as described previously [32 (link)]. Dual-luciferase assay reagents (Promega) were used to measure the activities of firefly luciferase and Renilla luciferase on an Infinite M200 PRO enzyme labeling instrument (Tecan, Hombrechtikon, Switzerland) as described [39 (link)]. All experiments were conducted with three independent replicates.
8
Dual Luciferase Assays for Arabidopsis and Loquat
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Dual luciferase assays were performed according to our previous reports (Yin et al., 2010 (link); Min et al., 2012 (link)). The promoters of the Arabidopsis and loquat lignin biosynthesis genes were amplified with the primers described in Supplementary Tables S3 and S4 , respectively. Full-length EjMYB1 and EjMYB2 were inserted into the pGreen II 0029 62-SK vector (SK), while the promoters were inserted into the pGreen II 0800-LUC vector. Details of vector information are given in Hellens et al. (2005) (link).
All the constructs were electroporated into Agrobacterium tumefaciens GV3101. The dual luciferase assays were performed with Nicotiana benthamiana leaves. Agrobacterium cultures were prepared with infiltration buffer (10mM MES, 10mM MgCl2, 150mM acetosyringone, pH 5.6) to an OD600 of 0.7–1.0. Agrobacterium culture mixtures of transcription factors (1ml) and promoters (100 µl) were infiltrated into tobacco leaves using needleless syringes. The tobacco plants were grown in a glasshouse with daylight extension to 16h. Three days after infiltration, firefly luciferase and Renilla luciferase were assayed using dual luciferase assay reagents (Promega). For each transcription factor–promoter interaction, three independent experiments were performed (at least five replicates in each experiments).
All the constructs were electroporated into Agrobacterium tumefaciens GV3101. The dual luciferase assays were performed with Nicotiana benthamiana leaves. Agrobacterium cultures were prepared with infiltration buffer (10mM MES, 10mM MgCl2, 150mM acetosyringone, pH 5.6) to an OD600 of 0.7–1.0. Agrobacterium culture mixtures of transcription factors (1ml) and promoters (100 µl) were infiltrated into tobacco leaves using needleless syringes. The tobacco plants were grown in a glasshouse with daylight extension to 16h. Three days after infiltration, firefly luciferase and Renilla luciferase were assayed using dual luciferase assay reagents (Promega). For each transcription factor–promoter interaction, three independent experiments were performed (at least five replicates in each experiments).
9
Dual-luciferase assay for AP2/ERF transcriptional regulation
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Transactivation activities of AP2/ERF on the target promoter were measured with dual-luciferase assays as previously reported (Yin et al., 2010 (link); Xu et al., 2014 (link)). Full-length CitERF71 sequences were amplified with the primers described in Supplementary Table S1 and were inserted into a pGreenII 0029 62-SK vector. The promoter of CitTPS16 (2437 bp) was constructed in the pGreenII 0800-LUC vector. All constructs were individually electroporated into Agrobacterium GV3101 and stored as glycerol stocks at –80 °C. Agrobacterium cultures were prepared with infiltration buffer (10 mM MES, 10 mM MgCl2, 150 mM acetosyringone, pH 5.6) to an OD600 of 0.75. The mixtures of transcription factors (1 ml) and promoters (100 μl) were infiltrated into tobacco leaves by needleless syringes. Nicotiana benthamiana plants were grown in a growth chamber with a light/dark cycle of 16:8 h, at 24 °C. Four-week-old plants were prepared for injection. Enzyme activities of firefly luciferase and renilla luciferase were assayed using dual-luciferase assay reagents (Promega), at 3 d after infiltration. For each transcription factor–promoter interaction, at least three independent experiments were performed, with four replicates in each experiment.
10
Dual-Luciferase Assay for Gene Expression
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Dual-luciferase assay was performed to evaluate the luciferase activity of the reporter constructs according to the instructions of dual-luciferase assay reagents (Promega, USA) with some modifications. In brief, HEK293 cells were seeded on a 24-well plate, and the cells were allowed to grow until 80% confluency. Cells were then transfected with TOPFlash or pmiR-GLO luciferase reporter plasmids together with linc-ROR expression vectors or miRNA mimics. The pRSV-β-galactoside (ONPG) vector was co-transfected into HEK293 cells as an internal control for normalization. The plate was placed into a PerkinElmer VictorTM X2 2030 multilabel reader (Waltham, USA) to measure the firefly luciferase activity, as well as the β-galactosidase activity. The ratio of firefly luciferase to β-galactosidase activity in each sample was revealed as a measurement of the normalized luciferase activity. All experiments were performed in triplicate.
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