| miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells. | |
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MedLine Citation:
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PMID: 22417692 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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MicroRNA-mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3' UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3' UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3' UTR-mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs. |
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Authors:
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Emmanuel Beillard; Siau Chi Ong; Antonis Giannakakis; Ernesto Guccione; Leah A Vardy; P Mathijs Voorhoeve |
Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2012-03-14 |
Journal Detail:
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Title: RNA (New York, N.Y.) Volume: 18 ISSN: 1469-9001 ISO Abbreviation: RNA Publication Date: 2012 May |
Date Detail:
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Created Date: 2012-04-18 Completed Date: 2012-06-12 Revised Date: 2013-05-20 |
Medline Journal Info:
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Nlm Unique ID: 9509184 Medline TA: RNA Country: United States |
Other Details:
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Languages: eng Pagination: 1091-100 Citation Subset: IM |
Affiliation:
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Department of Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore 169857, Singapore. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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3' Untranslated Regions Animals Argonaute Proteins / metabolism Base Sequence Cell Line Gene Expression Gene Order Genes, Reporter* Genetic Vectors Humans Luciferases / genetics, metabolism Mice MicroRNAs / metabolism* Molecular Sequence Data Poly A / chemistry Protein Biosynthesis RNA, Messenger / metabolism Retroviridae / genetics |
| Chemical | |
Reg. No./Substance:
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0/3' Untranslated Regions; 0/Argonaute Proteins; 0/MicroRNAs; 0/RNA, Messenger; 24937-83-5/Poly A; EC 1.13.12.-/Luciferases |
| Comments/Corrections | |
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