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Methods

Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis

¹Hubrecht Laboratory, 3584 CT Utrecht, The Netherlands; ²Department of Cardiology, HLCU Location, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands; ³Department of Nephrology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; ⁴Department of Physiology, Free University Medical Center, 1081 BT Amsterdam, The Netherlands; ⁵Divisionof Functional Genomics, Jichi Medical School, Kawachigun, Tochigi 329-0498, Japan

icroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently >400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.

-mail plasterk{at}niob.knaw.nl; fax 31-30-2516554.

rticle published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.5159906.

Supplemental material is available online at www.genome.org. All novel microRNAs described in this study are submitted to the central miRNA repository miRBase, maintained at the Wellcome Trust Sanger Institute, under the ID nos. listed in the Supplemental material.

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