Document Detail

RISC RNA sequencing for context-specific identification of in vivo microRNA targets.
MedLine Citation:
PMID:  21030712     Owner:  NLM     Status:  MEDLINE    
RATIONALE: MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo.
OBJECTIVE: To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs).
METHODS AND RESULTS: We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1645 mRNAs consistently targeted to mouse cardiac RISCs. We used this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing "seed" sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts.
CONCLUSIONS: RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context and is applicable to any tissue and any disease state.
Scot J Matkovich; Derek J Van Booven; William H Eschenbacher; Gerald W Dorn
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2010-10-28
Journal Detail:
Title:  Circulation research     Volume:  108     ISSN:  1524-4571     ISO Abbreviation:  Circ. Res.     Publication Date:  2011 Jan 
Date Detail:
Created Date:  2011-01-07     Completed Date:  2011-02-09     Revised Date:  2013-07-03    
Medline Journal Info:
Nlm Unique ID:  0047103     Medline TA:  Circ Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  18-26     Citation Subset:  IM    
Center for Pharmacogenomics, Department of Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.
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MeSH Terms
Gene Expression Profiling / methods*
Gene Expression Regulation / physiology
Mice, Transgenic
MicroRNAs* / biosynthesis,  genetics
Myocardium / metabolism*
RNA, Messenger / biosynthesis*,  genetics
RNA-Induced Silencing Complex* / genetics,  metabolism
Sequence Analysis, RNA / methods*
Grant Support
R01 HL059888-01A1/HL/NHLBI NIH HHS; R01 HL059888-02/HL/NHLBI NIH HHS; R01 HL059888-03/HL/NHLBI NIH HHS; R01 HL059888-04/HL/NHLBI NIH HHS; R01 HL059888-05/HL/NHLBI NIH HHS; R01 HL059888-06/HL/NHLBI NIH HHS; R01 HL059888-07/HL/NHLBI NIH HHS; R01 HL059888-08/HL/NHLBI NIH HHS; R01 HL059888-09A1/HL/NHLBI NIH HHS; R01 HL059888-09A1W1/HL/NHLBI NIH HHS; R01 HL059888-10/HL/NHLBI NIH HHS; R01 HL059888-11/HL/NHLBI NIH HHS; R01 HL059888-12/HL/NHLBI NIH HHS; R01 HL059888-13/HL/NHLBI NIH HHS; R01 HL080008-01A1/HL/NHLBI NIH HHS; R01 HL080008-02/HL/NHLBI NIH HHS; R01 HL080008-03/HL/NHLBI NIH HHS; R01 HL080008-04/HL/NHLBI NIH HHS; R01 HL080008-05/HL/NHLBI NIH HHS; R01 HL080008-06/HL/NHLBI NIH HHS; R01 HL087871-01/HL/NHLBI NIH HHS; R01 HL087871-02/HL/NHLBI NIH HHS; R01 HL087871-03/HL/NHLBI NIH HHS; R01 HL087871-04/HL/NHLBI NIH HHS; R01 HL087871-05/HL/NHLBI NIH HHS; R01 HL087871-06/HL/NHLBI NIH HHS; R01 HL59888/HL/NHLBI NIH HHS; RC2 HL102222/HL/NHLBI NIH HHS; RC2 HL102222-01/HL/NHLBI NIH HHS; RC2 HL102222-02/HL/NHLBI NIH HHS; UL1 RR024992/RR/NCRR NIH HHS; UL1 TR000448/TR/NCATS NIH HHS
Reg. No./Substance:
0/MicroRNAs; 0/RNA, Messenger; 0/RNA-Induced Silencing Complex
Comment In:
Circ Res. 2011 Jan 7;108(1):3-5   [PMID:  21212388 ]

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