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Nucleic Acids Research Advance Access originally published online on September 4, 2009
Nucleic Acids Research 2009 37(20):e137; doi:10.1093/nar/gkp715
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Nucleic Acids Research, 2009, Vol. 37, No. 20 e137
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A high throughput experimental approach to identify miRNA targets in human cells

Lu Ping Tan1, Erwin Seinen2, Gerben Duns3, Debora de Jong1, Ody C. M. Sibon2, Sibrand Poppema1, Bart-Jan Kroesen4, Klaas Kok3 and Anke van den Berg1,*

1Department of Pathology and Laboratory Medicine, 2Department of Cell Biology/Radiation and Stress Cell Biology, 3Department of Genetics and 4Department of Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands

*To whom correspondence should be addressed. Tel: +31 50 3611476; Fax: +31 50 3619107; Email: a.van.den.berg{at}path.umcg.nl

Received May 18, 2009. Revised July 16, 2009. Accepted August 14, 2009.

The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation—gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that ~40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.

The authors wish it to be known that, in their opinion, the second and third authors should be regarded as joint Second Authors.


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