Research Article
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Published Online: 29 March 2011

Observation of miRNA Gene Expression in Zebrafish Embryos by In Situ Hybridization to MicroRNA Primary Transcripts

Publication: Zebrafish
Volume 8, Issue Number 1

Abstract

MicroRNAs (miRNAs) add a previously unexpected layer to the post-transcriptional regulation of protein production. Although locked nucleic acids (LNAs) reveal the distribution of mature miRNAs by in situ hybridization (ISH) experiments in zebrafish and other organisms, high cost has restricted their use. Further, LNA probes designed to recognize mature miRNAs do not distinguish expression patterns of two miRNA genes that produce the same mature miRNA sequence. Riboprobes are substantially less expensive than LNAs, but have not been used to detect miRNA gene expression because they do not bind with high affinity to the short, 22-nucleotide-long mature miRNAs. To solve these problems, we capitalized on the fact that miRNAs are initially transcribed into long primary transcripts (pri-mRNAs). We show here that conventional digoxigenin-labeled riboprobes can bind to primary miRNA transcripts in zebrafish embryos. We tested intergenic and intronic miRNAs (miR-10d, miR-21, miR-27a, miR-126a, miR-126b, miR-138, miR-140, miR-144, miR-196a1, miR-196a2, miR-196a2b [miR-196c], miR-196b, miR-196b1b [miR-196d], miR-199, miR-214, miR-200, and miR-222) in whole mounts and some of these in histological sections. Results showed that pri-miRNA ISH provides an attractive and cost-effective tool to study miRNA expression by ISH. We use this method to show that miR-126a and miR-126b are transcribed in the caudal vasculature in the pattern of their neighboring gene ci116 or host gene egfl7, respectively, and that the chondrocyte miRNA mir-140 lies downstream of Sox9 in development of the craniofacial skeleton.

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cover image Zebrafish
Zebrafish
Volume 8Issue Number 1March 2011
Pages: 1 - 8
PubMed: 21288128

History

Published online: 29 March 2011
Published in print: March 2011
Published ahead of print: 2 February 2011

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Xinjun He
Institute of Neuroscience, University of Oregon, Eugene, Oregon.
Yi-Lin Yan
Institute of Neuroscience, University of Oregon, Eugene, Oregon.
April DeLaurier
Institute of Neuroscience, University of Oregon, Eugene, Oregon.
John H. Postlethwait
Institute of Neuroscience, University of Oregon, Eugene, Oregon.

Notes

Address correspondence to:John H. Postlethwait, Ph.D.Institute of NeuroscienceUniversity of OregonEugene, OR 97403E-mail: [email protected]

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No competing financial interests exist.

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