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2018 ; 9
(6
): ä Nephropedia Template TP
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MiRAR-miRNA Activity Reporter for Living Cells
#MMPMID29921790
Turk MA
; Chung CZ
; Manni E
; Zukowski SA
; Engineer A
; Badakhshi Y
; Bi Y
; Heinemann IU
Genes (Basel)
2018[Jun]; 9
(6
): ä PMID29921790
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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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