HOTAIR Promoter Luciferase Assay

HOTAIR enhancers and promoters were cloned into the luciferase reporter plasmid pGL3-Basic; see Supplementary Material, Table S7 for primers. The canonical HOTAIR promoter used in this study incorporated the previously described promoter region and E-box with additional bases either side (29 (link)). MCF7, T47D and MDA-MB-453 cells were transfected in antibiotic-free media with 700 ng of modified pGL3 promoter less reporter plasmids, 20 ng of Renilla and with 1μL of Lipofectamine® 2000 (Life Technologies, Grand Island, NY, USA). 48hours post transfection luciferase readings were measured using a DTX-880 luminometer and Dual-Glo® luciferase reporter kit (Promega, Madison, WI, USA), following the manufacturer’s recommended protocol. For oestrogen treatment and luciferase reporter levels of HOTAIR promoters and the HDE we followed the previously published method by Tan-Wong et al. (67 (link)).

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Milevskiy M.J., Al-Ejeh F., Saunus J.M., Northwood K.S., Bailey P.J., Betts J.A., McCart Reed A.E., Nephew K.P., Stone A., Gee J.M., Dowhan D.H., Dray E., Shewan A.M., French J.D., Edwards S.L., Clark S.J., Lakhani S.R, & Brown M.A. (2016). Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer. Human Molecular Genetics, 25(15), 3269-3283.

Publication 2016

Lipofectamine® 2000 DTX-880 luminometer Dual-Glo® luciferase reporter kit

Antibiotic Cells Lipofectamine 2000 Luciferase Mcf7 Oestrogen Pgl3 Plasmids Primers Promega Renilla Transfection

Corresponding Organization : University of Queensland

Other organizations : QIMR Berghofer Medical Research Institute, Indiana University Bloomington, Garvan Institute of Medical Research, Cardiff University, Queensland University of Technology, Royal Brisbane and Women's Hospital

Top 5 similar protocols

Variable analysis

independent variables

HOTAIR enhancers and promoters

dependent variables

Luciferase reporter levels
Luciferase readings

control variables

700 ng of modified pGL3 promoter less reporter plasmids
20 ng of Renilla
1μL of Lipofectamine® 2000

controls

Oestrogen treatment and luciferase reporter levels of HOTAIR promoters and the HDE following the previously published method by Tan-Wong et al. (67 (link))

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