Document Detail


miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells.
MedLine Citation:
PMID:  22417692     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Emmanuel Beillard; Siau Chi Ong; Antonis Giannakakis; Ernesto Guccione; Leah A Vardy; P Mathijs Voorhoeve
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-03-14
Journal Detail:
Title:  RNA (New York, N.Y.)     Volume:  18     ISSN:  1469-9001     ISO Abbreviation:  RNA     Publication Date:  2012 May 
Date Detail:
Created Date:  2012-04-18     Completed Date:  2012-06-12     Revised Date:  2013-06-26    
Medline Journal Info:
Nlm Unique ID:  9509184     Medline TA:  RNA     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1091-100     Citation Subset:  IM    
Affiliation:
Department of Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore 169857, Singapore.
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MeSH Terms
Descriptor/Qualifier:
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:
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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