Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, 35020 Legnaro (Padova), Italy.
Anal Biochem. 2013 Mar 1;434(1):60-6. doi: 10.1016/j.ab.2012.10.039. Epub 2012 Nov 10.
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.
微小 RNA（miRNA）影响发育的基本过程。在植物中，miRNA 调节器官发育、向开花的转变以及对非生物/生物胁迫的反应。为了了解 miRNA 的生物学作用，除了鉴定其靶向转录本外，还需要描述其表达的时空调控。许多方法已被用于通过组织解剖和 miRNA 分析来定义器官特异性 miRNA 的集合，但没有一种方法可以在原位杂交（ISH）提供的高分辨率下描述它们在组织和细胞中的分布。本文描述了一种使用 DIG 标记的 Zip 核酸（ZNA）寡核苷酸探针针对完整拟南芥幼苗内源 miRNA 的全菌原位杂交（ISH）方案的设置和优化。通过机器人化液体处理自动执行该程序的主要步骤，也已在该方案中实现，以获得最佳结果重现性，从而能够以高通量运行 ISH 实验。
