StarBase (http://starbase.sysu.edu.cn/ ) was utilized to predict the binding site of miR-16-5p to FOXK1. The mutant (mut) and wild-type (wt) sequences containing the binding sites of miR-16-5p to FOXK1 (mut-FOXK1 and wt-FOXK1) were designed and synthesized based on the predicted results and cloned into luciferase reporter vectors (pGL3-Basic). Thereafter, the designed sequences were co-transfected with miR-16-5p mimic (0, 150, 300 nM, GenePharma) into HEK293T cells, respectively. After that, cells received 20-min cell lysing with 100 μL cell lysis buffer on a shaking table at room temperature. Firefly luciferase activity or Renilla luciferase activity was measured after the exposure of cell suspension (50 μL) to luciferase reaction solution (50 μL, Promega, Madison, WI, USA) or Stop & Glo reagent (50 μL, Promega). The relative activity was calculated as the ratio of Firefly luciferase activity to Renilla luciferase activity. Renilla luciferase activity was regarded as an internal control. Three replicates were set for this test.
Luciferase reaction solution
Manufactured by Promega
Sourced in United States
Luciferase reaction solution is a laboratory reagent that facilitates the luciferase-luciferin reaction, which produces bioluminescence. The solution contains the necessary components for this enzymatic reaction to occur, enabling the detection and measurement of luciferase activity in various experimental settings.
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Lab products found in correlation
4 protocols using luciferase reaction solution
1
Validating miR-16-5p Binding Site in FOXK1
2
Validating miR-141-3p Interaction with EZH2
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Through the online database TargetScan (http://www.targetscan.org/vert_72/ ), the binding site of miR-141-3p and EZH2 was predicted. According to the predicted results, the wild sequence and mutant sequence (mut-EZH2, wt-EZH2) of the binding site were designed and synthesized, respectively. These sequences were inserted into the luciferase reporter gene vector (pGL3-Basic), respectively, and then cotransfected HEK293T cells with miR-141-3p mimic (0, 150 nM, 300 nM, GenePharma), respectively. After mixing well, 100 μl of cell lysis solution was added and the cells were allowed to fully lyse on a shaker for 20 min at room temperature. 50 μl of luciferase reaction solution (Promega, Madison WI, USA) was added to 50 μl of lysed cells to measure Firefly luciferase activity, while 50 μl of Stop & Glo reagent (Promega, USA) for detecting Renilla luciferase activity. Renilla luciferase activity was used as an internal reference, and the ratio of Firefly luciferase activity to Renilla luciferase activity was the relative activity of luciferase. Three replicates were set up for the experiment.
3
Validating miRNA-binding site interactions
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Wild and mutant sequences of the binding sites of F8 (wt-F8 and mut-F8) were designed and synthesized according to the online prediction. The sequence contained the 3′UTR fragment complementary to the target miRNA. Wt-F8 or mut-F8 was inserted into pGL3-Promoter vector and cotransfected with 50 nM hsa-miR-5581-3p mimic, hsa-miR-542-3p mimic or mimic NC together with Renilla luciferase reporter vector pRL-TK into HEK293T cells. The cells were added with 100 μl of lysis buffer and placed on a shaker for 20 min at room temperature. Suspension of the cell lysates (50 μl) was added with luciferase reaction solution (50 μl, Promega, Madison, WI, USA) before Firefly luciferase activity was measured. The suspension was then mixed with 50 μl of Stop&Glo reagent (Promega) to measure Renilla luciferase activity (internal reference). The ratio of firefly luciferase activity to Renilla luciferase activity was the relative luciferase activity.
4
HIF1A Regulates LINC02913 Promoter Activity
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The binding sites between HIF1A and LINC02913 promoter were predicted through the online database JASPAR (https://jaspar.genereg.net/analysis ). Based on the predicted results, the wild sequence and mutation sequence of the binding site (psicheck2-proLINC02913-WT, psicheck2-proLINC02913-MUT) were designed and synthesized respectively. The wild-type and mutant sequence of the binding sites were cloned into the luciferase reporter gene vector, respectively. Next, these constructed vectors were co-transfected with the control vector or HIF1A into HEK 293 T cells. Cells were added with 100 µL cell lysis buffer and placed on a shaking table at room temperature for 20 min for complete cell lysis. Next, 50 µL lysed cell suspension was added with 50 µL luciferase reaction solution (Promega, Madison WI, USA) for the determination of Firefly luciferase activity. Cells were added and mixed with 50 µL Stop&Glo reagent (Promega) for the determination of Renilla luciferase activity. Renilla luciferase activity was used as the internal reference. The relative luciferase activity was the ratio of Firefly luciferase activity to Renilla luciferase activity. Three replicates were set for the experiment. Primers used for plasmid construction are shown in Additional file 2 : Table S4.
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