title: Global identification of small RNA targets in plants by sequencing sliced ends of messenger RNAs.
abstract: Small RNAs (microRNAs and other classes of endogenous small interfering RNAs) play important roles in a wide variety of biological processes. However, integration of small RNAs in diverse biological networks relies on the confirmation of their RNA targets. In plants, miRNAs negatively regulate mRNA targets by guiding a cleavage in the complementary site that leaves a 3' polyadenylated RNA possessing monophosphate at its 5' end. This chapter describes a detailed step-by-step protocol for cloning such sliced 3' products in order to identify small RNA targets. Using this protocol, we have identified more than 150 small RNA targets in rice; some are conserved and others are non-conserved targets for rice small RNAs.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, USA.
authors.forename: Yong-Fang
authors.initials: YF
authors.lastname: Li
authors.forename: Ramanjulu
authors.initials: R
authors.lastname: Sunkar
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011786:Quality Control; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D018749:RNA, Plant / Q000235:genetics* / Q000302:isolation & purification; D058727:RNA, Small Untranslated / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D012333:RNA, Messenger; D018749:RNA, Plant; D058727:RNA, Small Untranslated