期刊
ANALYTICAL BIOCHEMISTRY
卷 434, 期 1, 页码 60-66出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.10.039
关键词
ISH; Whole mount ISH; miRNA; ZNA; Robotized liquid handling
资金
- European Project AUTO-SCREEN [LSHG-CT-2007-037897]
MicroRNAs (miRNAs) affect fundamental processes of development. In plants miRNAs regulate organ development, transition to flowering, and responses to abiotic/biotic stresses. To understand the biological role of miRNAs, in addition to identifying their targeted transcripts, it is necessary to characterize the spatiotemporal regulation of their expression. Many methods have been used to define the set of organ-specific miRNAs by tissue dissection and miRNA profiling but none of them can describe their tissue and cellular distribution at the high resolution provided by in situ hybridization (ISH). This article describes the setup and optimization of a whole-mount ISH protocol to target endogenous miRNAs on intact Arabidopsis seedlings using DIG-labeled Zip Nucleic Acid (ZNA) oligonucleotide probes. Automation of the main steps of the procedure by robotized liquid handling has also been implemented in the protocol for best reproducibility of results, enabling running of ISH experiments at high throughput. (C) 2012 Elsevier Inc. All rights reserved.
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