Top fop flash

Manufactured by Genechem

Sourced in China

The TOP/FOP-Flash is a specialized laboratory instrument designed for performing transient transfection assays. It combines a luminometer and an automatic injector to enable rapid and efficient analysis of gene expression in cell-based systems. The device's core function is to detect and quantify reporter gene activity, providing researchers with valuable data for their experimental investigations.

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Lab products found in correlation

Top fop flash, by Promega (3 mentions) Renilla reniformis, by Promega (3 mentions) Dual luciferase report assay system, by Promega (3 mentions) Dual luciferase reporter assay, by Promega (2 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Pgl3 basic luciferase reporter vector, by Promega (1 mentions) Prl tk plasmid, by Promega (1 mentions) Pgl4.17 basic vector, by Promega (1 mentions) Dual luciferase reporter assay kit, by Transgene (1 mentions) Dual luciferase reporter system, by Promega (1 mentions) Prl sv40 vector, by Promega (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Pmirglo dual luciferase vector, by Promega (1 mentions)

12 protocols using top fop flash

Dual-Luciferase Assay for miRNA-LUCAT1 Regulation

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Full-length LUCAT1 sequence of wild-type (WT) and mutant-type (MUT) miRNA binding site was constructed in Genechem Co., Ltd. (Shanghai, China). LUCAT1 WT/MUT were transfected into MCF-7 with miR-5582-3p mimic/NC mimic. Similarly, the binding sites for miR-5582-3p in the 3′-untranslated region (3′-UTR) sequence of TCF7L2 were obtained from Genechem. TCF7L2 3′-UTR WT/MUT were transfected into MCF-7 with miR-5582-3p mimic/NC mimic. The Dual-Luciferase Reporter Assay (Promega) was applied according to the manufacturer’s instructions. For the TOP/FOP-Flash assay, TOP/FOP-Flash (Genechem) was co-transfected into cells along with LUCAT1 silence or overexpression vector, miR-5582-5p mimic or inhibitor, and/or the miRNA control. The TOP/FOP-Flash values were normalized to the Renilla reniformis (Promega) reading and the TOP/FOP ratio was measured, as previously described [25 (link)]. Experiments were performed in triplicate.

Zheng A., Song X., Zhang L., Zhao L., Mao X., Wei M, & Jin F. (2019). Long non-coding RNA LUCAT1/miR-5582-3p/TCF7L2 axis regulates breast cancer stemness via Wnt/β-catenin pathway. Journal of Experimental & Clinical Cancer Research : CR, 38, 305.

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Cyr61 Promoter Luciferase Assay

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Cyr61 promoter was sub-cloned into pGL3-Basic luciferase reporter vector (Promega, Madison, WI, USA) and then transfected into cells with indicated transfection plasmids or reagents. The promoter sequences of Cyr61 was obtained from Ensembl (http://asia.ensembl.org/index.html) and provided in Supplementary Table 1. The pGL3 vector containing Cyr61 promoter sequence was co-transfected with shCtrl, sh/circ-GLI1 or sh/circ-GLI1 + pcDNA3.1/p70S6K2 into HEK293T cells that were seeded into 24-well plates at a density of 2 × 10⁴ cells per well. Forty-eight hours later, Dual-Luciferase Report Assay System (Promega) was used to monitor luciferase activity. For TOP/FOP-Flash analysis, HEK-293T cells were co-transfected with TOP/FOP Flash (Genechem, Shanghai, China) together with shCtrl, sh/circ-GLI1 or sh/circ-GLI1 + pcDNA3.1/p70S6K2. All samples were assayed in triplicate.

Chen J., Zhou X., Yang J., Sun Q., Liu Y., Li N., Zhang Z, & Xu H. (2020). Circ-GLI1 promotes metastasis in melanoma through interacting with p70S6K2 to activate Hedgehog/GLI1 and Wnt/β-catenin pathways and upregulate Cyr61. Cell Death & Disease, 11(7), 596.

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Dual-Luciferase Assay for CMTM7 and miR-182

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The promoter regions of CMTM7 and miR-182-5p were cloned into a pGL4.17-basic vector (Promega) and transfected into breast cancer cells along with their corresponding controls and PRL-TK plasmid (Promega). Similarly, TOP/FOP-Flash (Genechem) was co-transfected into cells with CMTM7 overexpression or silence vector. The Dual-Luciferase Reporter Assay Kit (Transgene) was utilized to detect Firefly and Renilla luciferase activities in accordance with instructions.

Chen Z.H., Tian Y., Zhou G.L., Yue H.R., Zhou X.J., Ma H.Y., Ge J., Wang X., Cao X.C, & Yu Y. (2023). CMTM7 inhibits breast cancer progression by regulating Wnt/β-catenin signaling. Breast Cancer Research : BCR, 25, 22.

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Wnt/β-catenin Pathway Activation Assay

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TOP/FOP Flash (Genechem) was co-transfected into MHCC97-H, HCCLM3, or Hep 3B cells along with indicated plasmids as needed. The luciferase activity of TOP Flash or FOP Flash was monitored via Dual Luciferase Report Assay System (Promega, Madison, WI, USA). The TOP/FOP ratio was then calculated to assess the activity of Wnt/β-catenin pathway. Experiments were conducted three times.

Qin M., Meng Y., Luo C., He S., Qin F., Yin Y., Huang J., Zhao H., Hu J., Deng Z., Qiu Y., Hu G., Pan H., Qin Z., Huang Z, & Yi T. (2021). lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/β-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12. Molecular Therapy. Nucleic Acids, 25, 37-52.

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Dual Luciferase Reporter Assay for SOX9 and FARSA-AS1

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TOP/FOP-Flash assay was carried out as described previously^{24 (link)}. In short, cells were cotransfected with TOP/FOP-Flash (Genechem) and different plasmids for SOX9 overexpression, SOX9 inhibition, or FARSA-AS1 inhibition. After normalizing to the Renilla luciferase activity, the TOP/FOP ratio was measured via dual luciferase reporter system (Promega).

Zhou T., Wu L., Ma N., Tang F., Yu Z., Jiang Z., Li Y., Zong Z, & Hu K. (2020). SOX9-activated FARSA-AS1 predetermines cell growth, stemness, and metastasis in colorectal cancer through upregulating FARSA and SOX9. Cell Death & Disease, 11(12), 1071.

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Evaluating LINC01278 Effects on Transcriptional Activity

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Cells were seeded in a 96-well plate, and the TOP/FOP-Flash (Genechem) was co-transfected into cells along with LINC01278 siRNA or overexpression plasmid. Luciferase activity was measured using the Promega Dual-Luciferase Reporter Assay System at 24 h after transfection. The luciferase activity was normalized to the Renilla luciferase activity.

Lin S., Zhang W., Shi Z., Tan L., Zhu Y., Li H, & Peng X. (2021). β-Catenin/LEF-1 transcription complex is responsible for the transcriptional activation of LINC01278. Cancer Cell International, 21, 380.

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WNT2B Regulation by LINC00665

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TOP/FOP‐Flash (Genechem) was co‐transfected into cells (5 × 10⁵) along with the transfection of sh‐NC, sh‐LINC00665 or sh‐LINC00665 + pcDNA‐WNT2B. The TOP/FOP‐Flash values were normalized to the Renilla reniformis (Promega).

Bai J., Zhang X., Jiang F., Shan H., Gao X., Bo L, & Zhang Y. (2022). A Feedback Loop of LINC00665 and the Wnt Signaling Pathway Expedites Osteosarcoma Cell Proliferation, Invasion, and Epithelial‐Mesenchymal Transition. Orthopaedic Surgery, 15(1), 286-300.

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Transcriptional Activity Assay of PDHB-AS

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This assay was implemented as previously described [24 (link)]. TOP/FOP-Flash (Genechem) was co-transfected into CC cells with pcDNA3.1-PDHB-AS or empty vector (pcDNA3.1). After transfection for 48 h, both firefly and Renila luciferase activities were measured. The luciferase activity of TOP/FOP-Flash was normalized to the Renilla luciferase activity. Experiments were performed in triplicate.

Chi C., Hou W., Zhang Y., Chen J., Shen Z., Chen Y, & Li M. (2023). PDHB-AS suppresses cervical cancer progression and cisplatin resistance via inhibition on Wnt/β-catenin pathway. Cell Death & Disease, 14(2), 90.

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Quantitative Analysis of Wnt/β-catenin Signaling

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When the Wnt/β-catenin pathway is activated, β-catenin is translocated to the nucleus, where in complexes with members of the TCF/LEF family of transcription regulators, activates transcription of TCF-responsive genes. TOP-Flash is a TCF-reporter plasmid, along with FOP-Flash enables quantitation of the Wnt/β-catenin signaling in cells transfected with these constructs. TOP/FOP-Flash (Genechem) was cotransfected into cells with NC mimics, miR-543 mimics, miR-543 + pcDNA3.1, miR-543 + MAPK1. The pRL-SV40 vector (Promega) that encodes renilla luciferase gene was used to standardize the transfection efficiency. Luciferase assay was conducted with the Dual Luciferase Assay System kit 36 h after transfection. Relative luciferase activity is reported as fold induction (TOP/FOP) after normalization for transfection efficiency. Each biological sample was run in triplicate and experiments were independently repeated three times.

Wang L., Liu D., Wei J., Yuan L., Zhao S., Huang Y., Ma J, & Yang Z. (2021). MiR-543 Inhibits the Migration and Epithelial-To-Mesenchymal Transition of TGF-β-Treated Endometrial Stromal Cells via the MAPK and Wnt/β-Catenin Signaling Pathways. Pathology and Oncology Research, 27, 1609761.

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Wnt Signaling Pathway Activation Assay

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Briefly, tumor cells (5 × 10⁴) were seeded into a 24-well plate, and TOP-Flash reporter plasmids and pTK-RL plasmids were transiently co-transfected into the cells using Lipofectamine 2000 (Invitrogen). After transfection for 48 hours, the Dual-Luciferase Reporter Assay (Promega) was applied according to the manufacturer’s instructions. For the TOP/FOP-Flash assay, TOP/FOP-Flash (Genechem) was co-transfected into cells along with HNRNPM depletion or overexpression, HNRNPM-ASO, MBD2a silence, FZD3 silence, or MBD2c overexpression, and/or control. The TOP/FOP-Flash values were normalized to the Renilla reniformis (Promega) reading and the TOP/FOP ratio was measured. Experiments were performed in triplicate.

Zhu G.Q., Wang Y., Wang B., Liu W.R., Dong S.S., Chen E.B., Cai J.L., Wan J.L., Du J.X., Song L.N., Chen S.P., Yu L., Zhou Z.J., Wang Z., Zhou J., Shi Y.H., Fan J, & Dai Z. (2022). Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma. Cellular and Molecular Gastroenterology and Hepatology, 13(5), 1413-1447.

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