RNA Detection and Visualization: Methods and Protocols

ISBN-13: 9781493958368 / Angielski / Miękka / 2016 / 497 str.

Jeffrey E. Gerst

Opis

This book presents methods and protocols for both fixed and live cells from everything from bacteria to mammalian models, offering detailed techniques from experts in the field. The text includes vital notes on troubleshooting and avoiding known pitfalls.

Spis treści

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 Colón-Ramos, and William Zerges

 

3. High Resolution Fluorescent In situ Hybridization in Drosophila

            Eric Lécuyer

 

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

            José Roberto Sotelo-Silveira, Aldo Calliari, Alejandra Kun, Victoria Elizondo, Lucía Canclini, and José 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 André 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

Blurb

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™ 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.