Luciferase Reporter Assay for miR-302d Target

Luciferase reporter assay was performed according to a previously defined protocol^{20 (link),61 (link)}. Briefly, cells were seeded into 24-well plates and transfected with 16 ng cytomegalovirus-Renilla (Promega), 20 pmol miR-302d mimic or NC mimic, and 800 ng CDKN1A^WT or CDKN1A^MU. Cells were collected 72 h post transfection for luciferase activities measurement using the dual luciferase system (Promega) with a GloMax-96 luminometer. Renilla luciferase activities were taken as internal standard indicators for transfection efficiency. Firefly luciferase activities were further normalized to Renilla luciferase activities.

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Jiang C., Xie P., Sun R., Sun X., Liu G., Ding S., Zhu M., Yan B., Liu Q., Chen X, & Zhao C. (2018). c-Jun-mediated microRNA-302d-3p induces RPE dedifferentiation by targeting p21Waf1/Cip1. Cell Death & Disease, 9(5), 451.

Publication 2018

cytomegalovirus-Renilla dual luciferase system GloMax-96 luminometer

Assay Cells Cytomegalovirus Firefly luciferase Luciferase Promega Renilla Renilla luciferase Transfection

Corresponding Organization :

Other organizations : Nanjing Medical University, Jiangsu Province Hospital, Zhengzhou Children's Hospital, Qilu Hospital of Shandong University, University of Sydney, Eye & ENT Hospital of Fudan University

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Variable analysis

independent variables

MiR-302d mimic or NC mimic
CDKN1AWT or CDKN1AMU

dependent variables

Firefly luciferase activities

control variables

Cells seeded into 24-well plates
Cells collected 72 h post transfection
Renilla luciferase activities (used as internal standard indicators for transfection efficiency)

controls

Positive control: Cytomegalovirus-Renilla (Promega)
Negative control: NC mimic

Annotations

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