To predict the target gene of AFAP1-AS1, we first performed a bioinformatics analysis. We identified potential genes and focused on miR-145 based on the RNAhybrid results. We then constructed vectors carrying the wild-type or mutated miR145 3′-untranslated region (3′-UTR), which are referred to as pmirGLO/AFAP1-AS1-3′UTR and pmirGLO/AFAP1-AS1-3′UTR Mut, respectively (the detailed construction protocol is described in the supplementary information). For the Luciferase reporter assay, MDA-MB-231 cells were seeded into 6-well plates at a density of 1 × 105/well and grown in 2 mL DMEM for 24 h to reach 70%–80% confluency. The medium was then replaced with 1 mL DMEM without antibodies, and the cells were transfected with each luciferase reporter gene (20 pmol) diluted in 50 μL Opti-MEM and 4 μL Lipofectamine 2000 (Invitrogen) for 24 h. The dual luciferase assay was performed using a Dual luciferase reporter gene detection kit (Cat. #RG009, Biyuntian, Jiangsu, China) according to the manufacturer’s instructions on a GloMax machine (Promega, Madison, WI, USA). The data were normalized to Renilla luciferase activity.
Glomax machine
Manufactured by Promega
Sourced in United States
The Glomax machine is a laboratory equipment designed for luminescence detection and analysis. It is used to measure light output from luciferase-based reporter assays, including bioluminescence and chemiluminescence. The Glomax machine provides accurate and sensitive luminescence detection for a variety of applications in life science research.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Cignal finder pathway reporter arrays, by Qiagen (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
5 protocols using glomax machine
1
Luciferase Assay to Identify AFAP1-AS1 Target
2
Pathway Activity Analysis in GBM Cells
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Analysis on cancer-related pathways activity was performed using the Cignal Finder Pathway Reporter Arrays (SA Biosciences, Fredrick, MD). GBM cells were seeded into a 96-well white plate and incubated overnight at 37°C. Transient transfection was conducted by adding plasmid construct of transcription factor-responsive reporter of each pathway and controls to cells and incubated overnight in a 37°C incubator. Then, cells were further incubated for 48 hours. Each transfection condition was carried in triplicates. After 48 hours, the changes in expression of each pathway in cells with or without EMP3 depletion were determined by measuring the generated firefly and Renilla luminescent signals using the Dual-Glo Luciferase Assay system (Promega, Madison, WI) on the Glomax machine (Promega, USA). The relative luciferase units were determined by dividing the firefly to Renilla luciferase activity ratio. The luciferase activity of scramble shRNA control was regarded as 100%.
3
Pathway Analysis of CEACAM19 Depletion in Cancer Cells
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Analysis on cancer-related pathways activity was performed using the Cignal Finder Pathway Reporter Arrays (SA Biosciences, Fredrick, MD, USA) as described previously.14 (link) Penl1 cells were seeded into a 96-well white plate and incubated overnight at 37°C. Transient transfection was conducted by adding plasmid construct of transcription factor-responsive reporter of each signaling pathway and controls to cells and incubated overnight in a 37°C incubator. Then, cells were further incubated for 48 hours. Each transfection condition was carried in triplicates. After 48 hours, the changes in expression of each pathway in cells with or without CEACAM19 depletion were determined by measuring the generated firefly and Renilla luminescent signals using the Dual-Glo Luciferase Assay system (Promega, Madison, WI, USA) on the Glomax machine (Promega). The relative luciferase units were determined by dividing the firefly to Renilla luciferase activity ratio. The luciferase activity of Scr shRNA control was regarded as 100%.
4
Luciferase Assay for p53 Activity
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The pGL3M-enhancer and p53-luc vectors were obtained from Tiangen (Beijing, China) and used to assess luciferase activity. The pGL3-enhancer vector contains an SV40-enhancer located downstream of luc+ and the poly A signal. It assisted in the verification of the functional promoter elements. This is why the presence of an enhancer will frequently result in the transcription of luc+ at higher levels. The p53-luc vector contained a p53 response element and a luciferase reporter gene at positions 32 nt~85 nt and 168 nt~1830 nt, respectively. For the luciferase reporter assay, VMRC-LCD cells were grown in 24-well plates and transfected with pcDNA3.1 and pcDNA3.1-H19, plus pGL3M-enhancer and p53-luc vectors using lipofectamine 2000 (Invitrogen) for 24 h according to the manufacturer’s instructions. Next, the total cellular protein was extracted using the RIPA buffer and quantified. The total protein was then subjected to the luciferase reporter assay using a luciferase dual assay kit from Promega (Madison, WI, USA) and the luciferase activity was measured using a Promega Glomax machine. The data are presented as the mean ± s.d. of triplicate data.
5
Dual-Luciferase Assay Protocol for Plant
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LUC and REN luciferase activity was detected using a dual‐luciferase reporter assay system (Promega, USA) on Glomax machine (Promega, USA) as previously described (Iglesias‐Ara et al., 2018 (link)). Briefly, the effector construct pEG101‐GmDPB and reporter construct pGreen‐pGmCAT1 were introduced into Agrobacterium strain GV3101 separately and then expressed in tobacco leaves by A. tumefaciens‐mediated transient transformation. Leaf samples were collected and ground to powder at 4 °C separately. Luciferase Assay Substrate was resuspended in Luciferase Assay BufferII to generate LARII and 50 × Stop Glo substrate was added into Stop Glo Buffer to generate Stop Glo Reagent. After measuring firely luciferase activity, 20 μL Stop Glo Reagent was added into the mix to Renilla luciferase activity. The firefly LUC activity was normalized to the REN activity. The experiment was replicated three times independently.
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