[Show abstract][Hide abstract] ABSTRACT: The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using Parallel Analysis of RNA Ends (PARE) data is lacking in B. distachyon.
B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner.
B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.
[Show abstract][Hide abstract] ABSTRACT: For the experimental analysis of miRNAs and other small RNAs in the 20-25 nucleotide (nt) size range, the first and most important step is the isolation of high-quality total RNA. Because RNA degradation products can mask or dilute the presence of true miRNAs, it is important when choosing a method that it efficiently extracts RNA from tissues in a manner that prevents degradation of RNA of both high and low molecular weight. In addition, the presence of polyphenols, polysaccharides, and secondary metabolites may render nucleic acids insoluble, and hinder the recovery of the miRNAs. Finally, and most importantly, the method chosen must be capable of retaining the small RNA component. In this chapter, we will present a set of total RNA isolation methods that can be used to maximize the recovery of high-quality RNA to be used in miRNA analysis for a large number of plant species and tissue types.