Chimeric RNA Methods and Protocols

This book provides numerous methods for identification, validation, and functional characterization of chimeric RNAs, herein described as any transcript which contains the nucleotide sequence of two distinct parental genes. Beginning with identification and characterization through bioinformatics tools, the book continues with experimental validation using RT-PCR, hybridization methods for RNA, and the use of mass spectrometry data for fusion protein validation, as well as functional and mechanistic studies of chimeric RNAs and translational applications. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Part I: Identification and Characterization

 

1. Prediction, Characterization, and In Silico Validation of Chimeric RNAs

            Sandeep Singh and Hui Li

 

2. Identification of Fusion Transcripts from Unaligned RNA-Seq Reads Using ChimeRScope

            Neetha Nanoth Vellichirammal, Abrar Albahrani, You Li, and Chittibabu Guda

 

3. Identification of Chimeric RNAs Using RNA-Seq Reads and Protein-Protein Interactions of Translated Chimeras

            Milana Frenkel-Morgenstern

 

4. Gene Fusion Discovery with INTEGRATE

            Jin Zhang and Christopher A. Maher

 

5. Case Study: Systematic Detection and Prioritization of Gene Fusions in Cancer by RNA-Seq: A DIY Toolkit

            Pankaj Vats, Arul M. Chinnaiyan, and Chandan Kumar-Sinha

 

Part II: Experimental Validation

 

6. Detection and Measurement of Chimeric RNAs by RT-PCR

            Justin Elfman and Hui Li

 

7. Detection of Group II Intron-Generated Chimeric mRNAs in Bacterial Cells

            Félix LaRoche-Johnston, Caroline Monat, and Benoit Cousineau

 

8. RNase Protection Assay

            Jianzhu Zhao, Jun Tang, Justin Elfman, and Hui Li

 

9. Validation of Chimeric Fusion Peptides Using Proteomics Data

            Sandeep Singh

 

10. NanoString nCounter Technology: High-Throughput RNA Validation

            Angela Goytain and Tony Ng

 

Part III: Functional and Mechanistic Studies

 

11. Knockdown of Chimeric RNA by RNAi

            Xinrui Shi, Fujun Qin, and Hui Li

 

12. Overexpression of Chimeric RNA by Retroviral Transduction

            Hao Wu and Hui Li

 

13. Separation of Nuclear and Cytoplasmic Fractions for Chimeric RNA Characterization

            Xinrui Shi, Fujun Qin, and Hui Li

 

14. Confirmation of Transcriptional Read-Through Events by RT-PCR

            Xinrui Shi, Fujun Qin, and Hui Li

 

15. Validating Gene Fusion as the Source of Chimeric RNAs

            Sachin Kumar Gupta, Jocelyn Duen-Ya Jea, and Laising Yen

 

Part IV: Translational Applications

 

16. Chimeric RNA and Exosomes-Based Liquid Biopsy

            Xiurong Ke, Xiao Xiong, Yusheng Lin, and Hao Zhang

 

17. SMaRT for Therapeutic Purposes

            Lisa M. Riedmayr

 

18. Case Study: Landscape of Chimeric RNAs in Bladder Cancer

            Dingjun Zhu and Hui Li

 

19. Case Study: The Recurrent Fusion RNA DUS4L-BCAP29 in Non-Cancer Human Tissues and Cells

            Yue Tang, Fangxia Guan, and Hui Li