期刊
NATURE PROTOCOLS
卷 9, 期 4, 页码 939-949出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.061
关键词
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资金
- Massachusetts General Hospital Executive Committee on Research Postdoctoral Fellowship
- US National Science Foundation [IOS-0843244]
- US National Institutes of Health [R01 GM60493, R01 GM70567]
Artificial miRNA (amiRNA) technology offers highly specific gene silencing in diverse plant species. The principal challenge in amiRNA application is to select potent amiRNAs from hundreds of bioinformatically designed candidates to enable maximal target gene silencing at the protein level. To address this issue, we developed the epitope-tagged protein-based amiRNA (ETPamir) screens, in which single or multiple potential target genes encoding epitope-tagged proteins are constitutively or inducibly coexpressed with individual amiRNA candidates in plant protoplasts. Accumulation of tagged proteins, detected by immunoblotting with commercial tag antibodies, inversely and quantitatively reflects amiRNA efficacy in vivo. The core procedure, from protoplast isolation to identification of optimal amiRNA, can be completed in 2-3 d. The ETPamir screens circumvent the limited availability of plant antibodies and the complexity of plant amiRNA silencing at target mRNA and/or protein levels. The method can be extended to verify predicted target genes for endogenous plant miRNAs.
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