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Nucleic Acids Research Advance Access originally published online on September 14, 2006
Nucleic Acids Research 2006 34(17):e115; doi:10.1093/nar/gkl653
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Nucleic Acids Research, 2006, Vol. 34, No. 17 e115
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Mouse microRNA profiles determined with a new and sensitive cloning method

Shuji Takada1, Eugene Berezikov2, Yoshihiro Yamashita1, Mariana Lagos-Quintana3, Wigard P. Kloosterman2, Munehiro Enomoto1, Hisashi Hatanaka1, Shin-ichiro Fujiwara1, Hideki Watanabe1, Manabu Soda1, Young Lim Choi1, Ronald H. A. Plasterk2, Edwin Cuppen2 and Hiroyuki Mano1,4,*

1 Division of Functional Genomics, Jichi Medical University 3311-1 Yakushiji, Shimotsukeshi, Tochigi 329-0498, Japan 2 Hubrecht Laboratory Uppsalalaan 8, Utrecht, The Netherlands 3 Laboratory of RNA Molecular Biology, Rockefeller University 1230 York Avenue, New York, NY 10021, USA 4 CREST, Japan Science and Technology Agency Saitama 332-0012, Japan

*To whom correspondence should be addressed. Tel: +81 285 58 7449; Fax: +81 285 44 7322; Email: hmano{at}jichi.ac.jp

Received August 8, 2006. Revised August 14, 2006. Accepted August 19, 2006.

MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling method, termed miRNA amplification profiling (mRAP), as well as its application both to mouse embryos at various developmental stages and to adult mouse organs. A total of 77 436 Small-RNA species was sequenced, with 11 776 of these sequences found to match previously described miRNAs. With the use of a newly developed computational prediction algorithm, we further identified 229 independent candidates for previously unknown miRNAs. The expression of some of these candidate miRNAs was confirmed by northern blot analysis and whole-mount in situ hybridization. Our data thus indicate that the total number of miRNAs in vertebrates is larger than previously appreciated and that the expression of these molecules is tightly controlled in a tissue- and developmental stage-specific manner.

The history dates have been corrected.


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