RNA Tagging: Methods and Protocols

Part I: In Situ Detection of RNA Molecules Using FISH Techniques

1. Tagging and Application of RNA Probes for Sequence-Specific Visualization of RNAs by Fluorescent In Situ Hybridization

            Thomas Dresselhaus and Andrea Bleckmann

2. Quantitative Fluorescence In Situ Hybridization Detection of Plant mRNAs with Single-Molecule Resolution

            Kun Huang, Mona Batish, Chong Teng, Alex Harkess, Blake C. Meyers, and Jeffrey L. Caplan

3. Visualization of Endoplasmic Reticulum-Associated mRNA in Mammalian Cells

            Jingze J. Wu and Alexander F. Palazzo

4. Simultaneous Detection of mRNA and Protein in S. cerevisiae by Single Molecule FISH and Immunofluorescence

            Evelina Tutucci and Robert H. Singer

Part II: In Vivo Imaging of RNA Transport and Localization

5. Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More

            Michael Ryckelynck

6. Visualization of Transiently Expressed mRNA in Plants Using MS2

            Eduardo José Peña and Manfred Heinlein

7. New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells

            Xavier Pichon, Marie-Cécile Robert, Edouard Bertrand, Robert H. Singer, and Evelina Tutucci

8. In Vivo Imaging of Mobile mRNAs in Plants Using MS2

            Kai-Ren Luo, Nien-Chen Huang, and Tien-Shin Yu

9. RNA Imaging with RNase-Inactivated Csy4 in Plants and Filamentous Fungi

            David Burnett, Alexander Lichius, and Jens Tilsner

Part III: Imaging and Analysis of RNA Uptake and Transport between Cells

10. Utilizing Potato Virus X to Monitor RNA Movement

            Zhiming Yu, Sung Ki Cho, Pengcheng Zhang, Yiguo Hong, and David J. Hannapel

11. A Protocol for Non-Biased Identification of RNAs Transferred between Heterologous Mammalian Cell Types Using RNA Tagging, Cell Sorting, and Sequencing

            Sandipan Dasgupta and Jeffrey E. Gerst

12. Synthesizing Fluorescently Labeled dsRNAs and sRNAs to Visualize Fungal RNA Uptake

            Rachael Hamby, Ming Wang, Lulu Qiao, and Hailing Jin

13. Labeling of dsRNA for Fungal Uptake Detection Analysis

            Matteo Galli, Jafargholi Imani, and Karl-Heinz Kogel

Part IV: Identification and Analysis of RNA-Binding Proteins

14. Using RNA Affinity Purification Followed by Mass Spectrometry to Identify RNA-Binding Proteins (RBPs)

            Mengge Shan and Brian D. Gregory

15. Plant Individual Nucleotide Resolution Crosslinking and Immunoprecipitation to Characterize RNA-Protein Complexes

            Tino Köster and Dorothee Staiger

16. A Single-Molecule RNA Mobility Assay to Identify Proteins that Link RNAs to Molecular Motors

            Varun Bhaskar, Min Jia, and Jeffrey A. Chao

17. Proximity-CLIP Provides a Snapshot of Protein-Occupied RNA Elements at Subcellular Resolution and Transcriptome-Wide Scale

            Daniel Benhalevy and Markus Hafner

18. Double-Stranded RNA Pull-Down to Characterize Viral Replication Complexes in Plants

            Marco Incarbone and Christophe Ritzenthaler

Part V: RNAs as Guides for Genome Editing and Imaging of Chromosome Loci

19. CRISPR Guide RNA Design Guidelines for Efficient Genome Editing

            Patrick Schindele, Felix Wolter, and Holger Puchta

20. Live Cell CRISPR Imaging in Plant Cells with a Telomere-Specific Guide RNA

21. Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular Beacon Hybrid Systems

            Xiaotian Wu, Yachen Ying, Shiqi Mao, Christopher J. Krueger, and Antony K. Chen

22. Using RNA Tags for Multicolor Live Imaging of Chromatin Loci and Transcription in Drosophila Embryos

            Hongtao Chen and Thomas Gregor

Part VI: Specific Techniques for the Analysis of RNA Functions

23. Integrated Genome-Scale Analysis and Northern Blot Detection of Retrotransposon siRNAs across Plant Species

            Marcel Bohrer, Bart Rymen, Christophe Himber, Aude Gerbaud, David Pflieger, Debbie Laudencia-Chingcuanco, Amy Cartwright, John Vogel, Richard Sibout, and Todd Blevins

24. Transfection of Small Non-Coding RNAs into Arabidopsis thaliana Protoplasts

            Stéphanie Lalande, Marjorie Chery, Elodie Ubrig, Guillaume Hummel, Julie Kubina, Angèle Geldreich, and Laurence Drouard

25. In Vivo Reporter System for miRNA-Mediated RNA Silencing

            Károly Fátyol

26. TRAP-SEQ of Eukaryotic Translatomes Applied to the Detection of Polysome-Associated Long Non-Coding RNAs

            Soledad Traubenik, Flavio Blanco, María Eugenia Zanetti, and Mauricio A. Reynoso

27. Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields Labeled RNA for Conformational Analysis and Imaging

            Manisha B. Walunj and Seergazhi G. Srivatsan