Main description:

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.

Contents:

1. Chromatin Immunoprecipitation Assay of Piwi in Drosophila

Hang Yin and Haifan Lin

2. Drosophila Germline Stem Cell for in vitro Analyses of PIWI-Mediated RNAi

Yuzo Niki, Takuya Sato, Takashi Yamaguchi, Ayako Saisho, Hiroshi Uetake and Hidenori Watanabe

3. RNAi and Overexpression of Genes in Ovarian Somatic Cells

Kuniaki Saito

4. Making piRNAs in vitro

Shinpei Kawaoka, Susumu Katsuma and Yukihide Tomari

5. A Framework for piRNA Cluster Manipulation

Ivan Olonikov, Adrien Le Thomas, and Alexe Aravin

6. Mass Spectrometric Analysis of 3'-End Methylation of piRNAs

Takeo Suzuki, Kenjyo Miyauchi, Yuriko Sakaguchi, and Tsutomu Suzuki

7. HITS-CLIP (CLIP-Seq) for Mouse Piwi Proteins

Anastassios Vourekas and Zissimos Mourelatos

8. Analysis of DNA Methylation in Mouse Testes

Satomi Kuramochi-Miyagawa, Kanako Kita-Kojima, Yusuke Shiromoto, Daisuke Ito, Hirotaka Koshima, and Toru Nakano

9. Analysis of Small RNA-Guided Endonuclease Activity in Endogenous Piwi Protein Complexes from Mouse Testes

Michael Reuter and Ramesh S. Pillai

10. Small RNA Library Construction from Minute Biological Samples

Jessica Matts, Yuliya Sytnikova, Guan-wei Chirn, Gabor Igloi and Nelson Lau

11. Analysis of sDMA Modifications of PIWI Proteins

Shozo Honda, Yoriko Kirino, and Yohei Kirino

12. Analyses of piRNA-Mediated Transcriptional Transposon Silencing in Drosophila: Nuclear Run-On Assay on Ovaries

Sergey Shpiz and Alla Kalmykova

13. Combined RNA/DNA Fluorescence in situ Hybridization on Whole-Mount Drosophila Ovaries

Sergey Shpiz, Sergey Lavrov, and Alla Kalmykova

14. Fast and Accurate Method to Purify Small Non-Coding RNAs from Drosophila Ovaries

Thomas Grentzinger and Severine Chambeyron

15. Isolation of Zebrafish Gonads for RNA Isolation

Hsin-Yi Huang and Rene Ketting

16. Small RNA Library Construction for High-Throughput Sequencing

Jon McGinn and Ben Czech

17. Analysis of Piwi-Loaded Small RNAs in Tetrahymena

Tomoko Noto, Henriette M. Kurth, and Kazufumi Mochizuki

18. Effective Gene Knockdown in the Drosophila Germline by Artificial miRNA-Mimicking siRNAs

Hailong Wang, Haidong Huang and Dahua Chen

19. Isolation and Bioinformatics Analyses of Small RNAs Interacting with Germ-Cell Specific Argonaute in Rice

Reina Komiya and Ken-ichi Nonomura