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Firefly and renilla dual luciferase assay kit

Manufactured by Biotium
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The Firefly and Renilla Dual Luciferase Assay kit is a laboratory tool designed to measure the activity of two different luciferase reporter enzymes simultaneously. The kit provides reagents and protocols for the quantitative analysis of firefly and Renilla luciferase activities in cell lysates or other biological samples.

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Lab products found in correlation

Passive lysis buffer (plb), by Biotium (1 mentions) Pgl4.75 hrluc cmv, by Promega (1 mentions) Synergy htx multi mode reader, by Agilent Technologies (1 mentions) Pmirglo vector, by Promega (1 mentions) Glomax 96 plate reader, by Promega (1 mentions) Dual glo luciferase assay, by Promega (1 mentions)

5 protocols using firefly and renilla dual luciferase assay kit

1

Quantifying TGFβ and BMP Pathway Activation

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HaCaT, HepG2 or A549 cells transiently transfected with the TGFβ/Smad-responsive CAGA12 promoter reporter construct (CAGA12-luc) were used for luciferase assays. The HaCaT CAGA12-Luc/TK-Renilla cells, which stably express the CAGA12-luc construct and the TK-Renilla luciferase reporter plasmid, were also used for luciferase assays. The BMP/Smad responsive promoter reporter (BRE2-luc) construct was transiently transfected to shControl or shTGFB2-AS1 HaCaT cells. The pCMV-β-gal construct, which encodes the β-galactosidase, was co-transfected with the transiently transfected promoter reporter plasmids for normalization of the firefly luciferase measurements. In the case of the HaCaT CAGA12-Luc/TK-Renilla cells, the firefly luciferase values were normalized to the Renilla-luc values. Luciferase reporter assays were performed using the Firefly and Renilla Dual Luciferase Assay kit from Biotium, Fremont CA, USA (BTIU30003-2). Relative normalized luciferase activity, which derives from average values from triplicate determinations, with standard deviations, is presented in the graphs. Each experiment was repeated at least twice.
Papoutsoglou P., Tsubakihara Y., Caja L., Morén A., Pallis P., Ameur A., Heldin C.H, & Moustakas A. (2019). The TGFB2-AS1 lncRNA Regulates TGF-β Signaling by Modulating Corepressor Activity. Cell Reports, 28(12), 3182-3198.e11.
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2

Luciferase Assay for IL-23 Binding Site

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The sequence of the putative PRINS binding site of IL-23 cDNA that was identified in silico was synthesized and inserted into the pmirGLO vector (Promega, Madison, WI, USA) between the Dra I and Xba I restriction sites, resulting in the pmirGLO-IL23BS construct (Supplementary Figure S4). HEK293 cells were transiently transfected by the PRINSpcDNA3.1 or pmirGLO plasmid as controls. Cells were also co-transfected with 0.5 μg pmirGLO-IL23BS or empty pmirGLO plasmid together with the PRINSpcDNA3.1 vector. In each transfection experiment, 0.025 μg renilla-luciferase-expressing pGL4.75 hRluc/CMV (Promega) plasmid was used as internal control. Cells were harvested after 24 h, washed with PBS (phosphate-buffered saline) and lysed with passive lysis buffer (Biotium, Hayward, CA, USA), and luciferase activities were measured using the Firefly and Renilla Dual Luciferase Assay Kit (Biotium) and SYNERGY/HTX Multi-Mode reader (Bio Tek Instruments, Winooski, VT, USA), according to the manufacturer’s instructions. The luciferase activity derived from the pmirGLO plasmids was normalized to the renilla-luciferase activity.
Kelemen E., Ádám É., Sági S.M., Göblös A., Kemény L., Bata-Csörgő Z., Széll M, & Danis J. (2022). Psoriasis-Associated Inflammatory Conditions Induce IL-23 mRNA Expression in Normal Human Epidermal Keratinocytes. International Journal of Molecular Sciences, 23(1), 540.
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3

Luciferase Assays for miRNA Regulation

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Luciferase assays in HaCaT or PC3U cells transiently transfected with the CAGA12-luciferase promoter reporter and siRNA pools, pcDNA3-MIR100HG, or miRNA mimics were performed using the firefly and renilla dual-luciferase Assay kit (Biotium, Fremont, CA, USA) as described [12 (link)]. Relative normalized luciferase activity is expressed as averages from triplicate determinations, with standard deviations. Each experiment was repeated at least twice.
Papoutsoglou P., Rodrigues-Junior D.M., Morén A., Bergman A., Pontén F., Coulouarn C., Caja L., Heldin C.H, & Moustakas A. (2021). The noncoding MIR100HG RNA enhances the autocrine function of transforming growth factor β signaling. Oncogene, 40(21), 3748-3765.
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4

Promoter Reporter Assay in A549 Cells

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Promoter reporter assays were carried out in 24-well plates using A549 parental cells as previously described11 (link)15 (link). Briefly, cells were co-transfected with Firefly luciferase reporter construct and Renilla luciferase control vector pGL4.10 (Promega). Firefly and Renilla luciferase values were measured 48 hours after transfection using Firefly and Renilla Dual Luciferase Assay Kit (Biotium #30005-1) or Dual-Glo luciferase assay (Promega #PAE2920) on a GloMax 96 plate reader (Promega). Renilla luciferase signals were normalized to Firefly luciferase signals.
Wood L.W., Cox N.I., Phelps C.A., Lai S.C., Poddar A., Talbot C J.r, & Mu D. (2016). Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome. Scientific Reports, 6, 19857.
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5

Epithelial Promoter Regulation Assay

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Py2T, A549, EpRas or MCF10A MII cells transiently transfected with the epithelial gene promoter reporter constructs (E‐cadherin‐luc, Esrp2‐luc, miR‐200s‐luc and CAGA9‐luc) were used for luciferase assays. TK‐Renilla luciferase reporter plasmid (pGL4.74, Promega, Madison, Wisconsin, USA) was co‐transfected with the transiently transfected promoter reporter plasmids for normalization of the firefly luciferase measurements. Luciferase reporter assays were performed using the Firefly and Renilla Dual Luciferase Assay kit (Biotium, Fremont CA, USA, Cat# BTIU30003‐2). Relative normalized luciferase activity from triplicate determinations derived average values with standard deviations and is presented in the graphs. Each experiment was repeated at least three times.
Tsubakihara Y., Ohata Y., Okita Y., Younis S., Eriksson J., Sellin M.E., Ren J., ten Dijke P., Miyazono K., Hikita A., Imamura T., Kato M., Heldin C, & Moustakas A. (2022). TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition. Molecular Oncology, 16(12), 2330-2354.
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