Main description:

With its complex and extensively regulated metabolism, the study of the RNA lifecycle demands tools that allow for the localization of RNAs to be observed either in an in situ setting or, preferably, under in vivo conditions. In RNA Detection and Visualization: Methods and Protocols, the best and brightest investigators provide an up-to-date and in-depth description of basic methods and protocols used for detecting and visualizing mRNAs in both fixed and live cells, from bacteria to mammals. For novices and experts alike, this mix of classic in situ hybridization and advanced live imaging techniques, cell fractionation and affinity purification procedures, and bioinformatics tools gives researchers the most complete and extensive array of research aids possible. As a volume written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and expert tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, RNA Detection and Visualization: Methods and Protocols offers well-honed techniques in order to inspire researchers around the world to further our knowledge of the vital biological significance of RNA.

Contents:

Part I: Visualizing mRNAs In situ

1. Single Molecule Imaging of RNA In situ

Mona Batish, Arjun Raj and Sanjay Tyagi

2. FISH and Immunofluorescence Staining in Chlamydomonas

James Uniacke, Daniel Colon-Ramos, and William Zerges

3. High Resolution Fluorescent In situ Hybridization in Drosophila

Eric Lecuyer

4. Localization and Anchorage of Maternal mRNAs to Cortical Structures of Ascidian Eggs and Embryos Using High Resolution In situ Hybridization

Alexandre Paix, Janet Chenevert, and Christian Sardet

5. Visualization of mRNA Localization in Xenopus Oocytes

James A. Gagnon and Kimberly L. Mowry

6. Visualization of mRNA Expression in the Zebrafish Embryo

Yossy Machluf and Gil Levkowitz

7. High-Resolution Fluorescence In situ Hybridization to Detect mRNAs in Neuronal Compartments In vitro and In vivo

Sharon A. Swanger, Gary J. Bassell, and Christina Gross

8. Localization of mRNA in Vertebrate Axons Using In situ Hybridization

Jose Roberto Sotelo-Silveira, Aldo Calliari, Alejandra Kun, Victoria Elizondo, Lucia Canclini, and Jose Roberto Sotelo

Part II: Visualizing mRNAs In vivo Using Molecular Probes or Reconstituted Fluorescent Proteins

9. Tiny Molecular Beacons for In vivo mRNA Detection

Diana P. Bratu, Irina E. Catrina, and Salvatore A.E. Marras

10. Delivery of Molecular Beacons for Live-Cell Imaging and Analysis of RNA

Antony K. Chen, Won Jong Rhee, Gang Bao, and Andrew Tsourkas

11. Genetically-Encoded Fluorescent Probes for Imaging Endogenous mRNA in Living Cells

Takeaki Ozawa and Yoshio Umezawa

12. Visualization of Induced RNA in Single Bacterial Cells

Azra Borogovac and Natalia E. Broude

Part III: Visualizing mRNAs In vivo Using Aptamers and Intact Fluorescent Proteins

13. Visualizing mRNAs in Fixed and Living Yeast Cells

Franck Gallardo and Pascal Chartrand

14. In vivo Visualization of RNA Using the U1A-Based Tagged RNA System

Sunglan Chung and Peter A. Takizawa

15. Visualizing Endogenous mRNAs in Living Yeast Using m-TAG, a PCR-Based RNA Aptamer Integration Method, and Fluorescence Microscopy

Liora Haim-Vilmovsky and Jeffrey. E. Gerst

16. Imaging mRNAs in Living Mammalian Cells

Sharon Yunger and Yaron Shav-Tal

17. Using the mRNA-MS2/MS2CP-FP System to Study mRNA Transport during Drosophila Oogenesis

Katsiaryna Belaya and Daniel St. Johnston

Part IV: Use of Cell Fractionation to Demonstrate the Sub-Cellular Localization of RNA

18. Genome-Wide Analysis of RNA Extracted from Isolated Mitochondria

Erez Eliyahu, Daniel Melamed, and Yoav Arava

19. Analyzing mRNA Localization to the Endoplasmic Reticulum via Cell Fractionation

Sujatha Jagannathan, Christine Nwosu, and Christopher V. Nicchitta

20. Isolation of mRNAs Encoding Peroxisomal Proteins from Yeast Using a Combined Cell Fractionation and Affinity Purification Procedure

Gadi Zipor, Cecile Brocard, and Jeffrey. E. Gerst

21. Profiling Axonal mRNA Transport

Dianna E. Willis and Jeffery L. Twiss

22. RNA Purification from Tumor Cell Protrusions Using Porous Polycarbonate Filters

Jay Shankar and Ivan R. Nabi

Part V: Affinity Purification of mRNAs and the Identification of Trans-Acting Factors

23. RNA-Binding Protein Immunopurification-Microarray (RIP-Chip) Analysis to Profile Localized RNAs

Alessia Galgano and Andre P. Gerber

24. RaPID: An Aptamer-Based mRNA Affinity Purification Technique for the Identification of RNA and Protein Factors Present in Ribonucleoprotein Complexes

Boris Slobodin and Jeffrey E. Gerst

25. RIP: An mRNA Localization Technique

Sabarinath Jayaseelan, Francis Doyle, Salvatore Currenti, and Scott Tenenbaum

26. The Dual Use of RNA Aptamer Sequences for Affinity Purification and Localization Studies of RNAs and RNA-Protein Complexes

Scott C. Walker, Paul D. Good, Theresa A. Gipson, and David R. Engelke

Part VI: Use of Bioinformatics to Identify Cis-Acting Motifs and Structures in RNAs

27. Identifying and Searching for Conserved RNA Localization Signals

Russell S. Hamilton and Ilan Davis

28. Computational Prediction of RNA Structural Motifs Involved in Post Transcriptional Regulatory Processes

Michal Rabani, Michael Kertesz, and Eran Segal