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This volume provides a collection of methods and protocols detailing extracellular RNA and hopes to be a useful resource for those interested in this exciting and emerging field. Chapters are divided into four sections covering an overview of the field, approaches for the isolation of exRNA, protocols for detection and quantitation of exRNA, and approaches that can be useful for studies to evaluate potential therapeutic applications. 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 tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Extracellular RNA: Methods and Protocols aims to ensure successful results in the further study of this vital field.

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1. Extracellular RNAs - A New Awareness of Old Perspectives

Noah Sadik, Lilian Cruz, Alessandra Gurtner, Rodosthenis S. Rodosthenous , Sophie A. Dusoswa, Olivia Ziegler, Thomas Sebastiaan Van Solinge, Zhiyun Wei, Ane Miren Salvador Garicano, Bence Gyorgy, Marike Broekman, and Leonora Balaj

2. Overview of Protocols for Studying Extracellular RNA and Extracellular Vesicles

Julia Small, Roger Alexander, and Leonora Balaj

3. Extracellular RNA Isolation from Cell Culture Supernatant

Sawen Bakr., Bridget Simonson, Kirsty M. Danielson, and Saumya Das

4. Use of a Hollow Fiber Bioreactor to Collect Extracellular Vesicles from Cells in Culture

Irene K. Yan, Neha Shukla, David A. Borrelli, and Tushar Patel

5. Isolation of Extracellular RNA from Serum/Plasma

Justyna Filant, Parham Nejad, Anu Paul, Bridget Simonson, Srimeenakshi Srinivasan, Xuan Zhang, Leonora Balaj, Saumya Das, Roopali Gandhi, Louise C. Laurent, and Anil K. Sood

6. Isolation of Extracellular RNA from Bile

Irene K. Yan, Valentin X. Berdah, and Tushar Patel

7. Cushioned-Density Gradient Ultracentrifugation (C-DGUC): a Refined and High Performance Method for the Isolation, Characterization & Use of Exosomes

Kang Li, David K. Wong, King Yeung Hong, and Robert L. Raffai

8. Magnetic Particle-Based Immunoprecipitation of Nanoscale Extracellular Vesicles from Biofluids

Pete Heinzelman

9. Enrichment of Extracellular Vesicle Subpopulations via Affinity Chromatography

Michelle E. Hung, Stephen B. Lenzini, Devin M. Stranford, and Joshua N. Leonard

10. Detection and Analysis of Non-vesicular Extracellular RNA

Juan Pablo Tosar and Alfonso Cayota

11. Isolation of Plasma Lipoproteins as a Source of Extracellular RNA

Kang Li, David K. Wong, Fu Sang Luk, Roy Y. Kim, and Robert L. Raffai

12. Droplet Digital PCR for Quantitation of Extracellular RNA

Irene K Yan, Rishabh Lohray, and Tushar Patel

13. Preparation of Small RNA NGS Libraries from Biofluids

Alton Etheridge, Kai Wang, David Baxter, and David Galas

14. Multiplexed Detection and Quantitation of Extracellular Vesicle RNA Expression using NanoString

Neha Shukla, Irene K. Yan, and Tushar Patel

15. Milk-derived Extracellular Vesicles for Therapeutic Delivery of Small Interfering RNAs

Akiko Matsuda and Tushar Patel

16. Loading of Extracellular Vesicles with Hydrophobically Modified siRNAs

Marie-Cécile Didiot, Reka A. Haraszti, Neil Aronin, and Anastasia Khvorova

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