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MiRAR-miRNA Activity Reporter for Living Cells.
Turk, Matthew A; Chung, Christina Z; Manni, Emad; Zukowski, Stephanie A; Engineer, Anish; Badakhshi, Yasaman; Bi, Yumin; Heinemann, Ilka U.
Afiliación
  • Turk MA; Department of Biochemistry, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada. mturk5@uwo.ca.
  • Chung CZ; Department of Biochemistry, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada. cchung88@uwo.ca.
  • Manni E; Department of Biochemistry, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada. emanni@uwo.ca.
  • Zukowski SA; Department of Biochemistry, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada. szukowsk@uwo.ca.
  • Engineer A; Department of Physiology and Pharmacology, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada. aengine@uwo.ca.
  • Badakhshi Y; Department of Biochemistry, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada. ybadakhs@uwo.ca.
  • Bi Y; Department of Biochemistry, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada. ybi@uwo.ca.
  • Heinemann IU; Department of Biochemistry, The University of Western Ontario, 1151 Richmond Street, London, ON N6A 5C1, Canada. ilka.heinemann@uwo.ca.com.
Genes (Basel) ; 9(6)2018 Jun 19.
Article en En | MEDLINE | ID: mdl-29921790
microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Genes (Basel) Año: 2018 Tipo del documento: Article País de afiliación: Canadá Pais de publicación: Suiza
Texto completo
Añadir a Mi BVS
Imprimir
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PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Genes (Basel) Año: 2018 Tipo del documento: Article País de afiliación: Canadá Pais de publicación: Suiza