XI, 249 p. 48 illus., 23 illus. in color. online resource
SUMMARY
PIWI-interacting RNAs (piRNAs) are the third and most-recently discovered group of silencing-inducible small RNAs in animals. PIWI-Interacting RNAs: Methods and Protocols provides the most recent methods and protocols for studying piRNAs in the gonads of a wide range of species, as well as in any other organs where piRNAs may be detected. Comprehensive high-throughput sequencing analysis of piRNAs in embryos, testes and ovaries of D. melanogaster, as well as in mouse and rat testes, has raised the profile of piRNAs and thus further accelerated piRNA studies. In addition to C. elegans, other model species such as Drosophila melanogaster, Arabidopsis thaliana and mice, along with cultured cell lines such as HeLa and Drosophila Schneider 2 (S2), and other such organisms have been used to address the fundamental questions of the biogenesis and functions of RNAi-triggering small non-coding RNAs. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, PIWI-Interacting RNAs: Methods and Protocols seeks to serve both established researchers and newcomers to the field to progress towards the ultimate goal of understanding the mechanisms and actions of piRNAs
CONTENT
Chromatin Immunoprecipitation Assay of Piwi in Drosophila -- Drosophila Germline Stem Cell for in vitro Analyses of PIWI-Mediated RNAi -- RNAi and Overexpression of Genes in Ovarian Somatic Cells -- Making piRNAs in vitro -- A Framework for piRNA Cluster Manipulation -- Mass Spectrometric Analysis of 3’-End Methylation of piRNAs -- HITS-CLIP (CLIP-Seq) for Mouse Piwi Proteins -- Analysis of DNA Methylation in Mouse Testes -- Analysis of Small RNA-Guided Endonuclease Activity in Endogenous Piwi Protein Complexes from Mouse Testes -- Small RNA Library Construction from Minute Biological Samples -- Analysis of sDMA Modifications of PIWI Proteins -- Analyses of piRNA-Mediated Transcriptional Transposon Silencing in Drosophila: Nuclear Run-On Assay on Ovaries -- Combined RNA/DNA Fluorescence in situ Hybridization on Whole-Mount Drosophila Ovaries -- Fast and Accurate Method to Purify Small Non-Coding RNAs from Drosophila Ovaries -- Isolation of Zebrafish Gonads for RNA Isolation -- Small RNA Library Construction for High-Throughput Sequencing -- Analysis of Piwi-Loaded Small RNAs in Tetrahymena -- Effective Gene Knockdown in the Drosophila Germline by Artificial miRNA-Mimicking siRNAs -- Isolation and Bioinformatics Analyses of Small RNAs Interacting with Germ-Cell Specific Argonaute in Rice
