Extracellular RNA: Methods and Protocols

215 Pages·2018·5.128 MB·English

by  Tushar Patel (eds.)

Methods in
Molecular Biology 1740
Tushar Patel Editor
Extracellular
RNA
Methods and Protocols
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
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Extracellular RNA
Methods and Protocols
Edited by
Tushar Patel
Department of Transplantation, Mayo Clinic, Jacksonville, FL, USA
Editor
Tushar Patel
Department of Transplantation
Mayo Clinic
Jacksonville, FL, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-7651-5 ISBN 978-1-4939-7652-2 (eBook)
https://doi.org/10.1007/978-1-4939-7652-2
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Preface
Extracellular RNAs (exRNAs), RNA molecules found outside the cellular environment, are
rapidly garnering attention as signaling mediators involved in intercellular communication and
also for their potential as disease biomarkers or therapeutics. Cell-to-cell communication is a
central component of cellular and tissue homeostasis, and development. Perturbation of these
processes can contribute to the pathogenesis or progression of disease. RNA molecules can be
released from cells but avoid degradation by incorporation within membrane bound vesicles
(extracellular vesicles) or by association with lipids or proteins. Retention of their structural
integrity contributes to retention of their biological effects. There are several different types of
exRNA, with specificity based on their cells of origin and evidence of selective release into extra-
cellular vesicles. They are found in the circulation and also in several biological fluids. Recognition
of their diverse functional involvement in cellular processes and participation in cell and tissue
development have all contributed to a rapidly growing interest in the field of exRNA.
The study of exRNA has been facilitated by the development of methods for the isola-
tion and analysis of exRNA. Many of the methods and protocols in this book have been
developed by groups supported by the Office of the NIH Director supported Common
Fund program on Extracellular RNA. The resources and initiatives supported by this pro-
gram have truly been a catalyst and galvanized research in exRNA.
The book is divided into four main sections, each of which contains contributions from
expert researchers in the field, and provides an authoritative and practical guide to the study
of exRNA. This collection of methods and protocols will serve as a useful resource for those
interested in this exciting and emerging field.
The first section provides an overview of the field. Chapter 1 starts with a concise and
up-to-date review of exRNA research, whereas Chapter 2 provides an overview that guides
the reader toward the use of appropriate protocols for isolation of exRNA, or extracellular
vesicles and their associated RNA content. These chapters provide the context and back-
ground for subsequent chapters that describe methods and protocols for isolation of exRNA.
The second section focuses on approaches for the isolation of exRNA. Chapters 3 and 4
describe methods for isolation of exRNA and RNA associated with extracellular vesicles from
cells in culture. An approach for the large-scale production of extracellular vesicles that pro-
vides greater exRNA yield is described in Chapter 4. An overview of standard approaches for
the isolation of extracellular vesicles from body fluids such as serum, plasma, or bile is pro-
vided in Chapters 5 and 6. These methods could be adopted for use in other body fluids, but
with modifications that depend on the nature, protein content, and composition of exRNA
within each fluid. Chapters 7–9 provide more specialized and refined approaches for separa-
tion of extracellular vesicles such as cushioned density gradient ultracentrifugation, magnetic
or affinity chromatography. These may be particularly useful for specific downstream applica-
tions and can be adapted for greater selectivity. Other than RNA present within extracellular
vesicles, exRNA can be associated with lipoproteins or other proteins. Protocols for the
isolation of such nonvesicular RNA are described in Chapters 10 and 11.
The third section outlines protocols for the detection and quantitation of exRNA. The
extent to which RNA content within extracellular vesicles reflects either their cellular origin
v
vi Preface
or underlying disease state will determine their potential application as biomarkers. The use
of exRNA as disease biomarkers requires reliable methods for their detection and quantita-
tion. The three chapters in this section discuss methods for the analysis of exRNA. Sensitivity
of detection is a major challenge in the field of exRNA because approaches such as Northern
blot or quantitative real-time PCR are often limited by the small amounts of exRNA pres-
ent in body fluids or within extracellular vesicles. Chapters 12–14 outline approaches for
the use of digital PCR, the optimization of approaches for preparation of libraries for next-
generation sequencing, and the use of nanostring for detection and analysis of exRNA.
The last section describes approaches that can be useful for studies to evaluate potential
therapeutic applications. Due to their important roles in intercellular communication,
extracellular vesicles have been evaluated as carriers to deliver therapeutic agents across
biological membranes. Chapters 15 and 16 outline methods for the use of extracellular
vesicles to deliver therapeutic RNA molecules.
The range of topics covered in this book will be of interest to scientists and researchers,
teachers and students, biotechnology companies and entrepreneurs interested in this rap-
idly emerging field of exRNA research. The sections are inter-related and are not just col-
lections of independent protocols. Thus, the refinement of methods for isolation of vesicular
or nonvesicular exRNA will complement the approaches for detection and quantitation.
Likewise, the application of appropriate methods for isolation of extracellular vesicles will
support approaches for their use in therapeutic applications. Nevertheless, individual
nuances are also reflected within the protocols, in order to provide a useful guide.
In closing, we would like to thank all of the authors for their outstanding and valuable
contributions, members of the NIH Common Fund Program on Extracellular RNA for
their support and guidance, Caitlyn Foerst for her expert administrative assistance, and for
all those involved in the production of the book. We hope that the reader will find this col-
lection valuable and that the use of the methods and protocols within this book will further
enhance the field of exRNA research.
Jacksonville, FL, USA Tushar Patel
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Extracellular RNAs: A New Awareness of Old Perspectives . . . . . . . . . . . . . . . . . 1
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Julia Small, Roger Alexander, and Leonora Balaj
3 Extracellular RNA Isolation from Cell Culture Supernatant . . . . . . . . . . . . . . . . 23
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Irene K. Yan, Neha Shukla, David A. Borrelli, and Tushar Patel
5 Isolation of Extracellular RNA from Serum/Plasma . . . . . . . . . . . . . . . . . . . . . . 43
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
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,
and Use of Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Kang Li, David K. Wong, King Yeung Hong, and Robert L. Raffai
8 Magnetic Particle-Based Immunoprecipitation of Nanoscale
Extracellular Vesicles from Biofluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Pete Heinzelman
9 Enrichment of Extracellular Vesicle Subpopulations Via Affinity
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Michelle E. Hung, Stephen B. Lenzini, Devin M. Stranford,
and Joshua N. Leonard
10 Detection and Analysis of Non-vesicular Extracellular RNA . . . . . . . . . . . . . . . . 125
Juan Pablo Tosar and Alfonso Cayota
11 Isolation of Plasma Lipoproteins as a Source of Extracellular RNA . . . . . . . . . . . 139
Kang Li, David K. Wong, Fu Sang Luk, Roy Y. Kim, and Robert L. Raffai
12 Droplet Digital PCR for Quantitation of Extracellular RNA . . . . . . . . . . . . . . . . 155
Irene K. Yan, Rishabh Lohray, and Tushar Patel
vii
viii Contents
13 Preparation of Small RNA NGS Libraries from Biofluids . . . . . . . . . . . . . . . . . . 163
Alton Etheridge, Kai Wang, David Baxter, and David Galas
14 Multiplexed Detection and Quantitation of Extracellular Vesicle RNA
Expression Using NanoString . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Neha Shukla, Irene K. Yan, and Tushar Patel
15 Milk-derived Extracellular Vesicles for Therapeutic Delivery of Small
Interfering RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Akiko Matsuda and Tushar Patel
16 Loading of Extracellular Vesicles with Hydrophobically Modified siRNAs . . . . . 199
Marie-Cecile Didiot, Reka A. Haraszti, Neil Aronin,
and Anastasia Khvorova
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Contributors
RogeR AlexAndeR • Pacific Northwest Diabetes Research Institute, Seattle, WA, USA
neil ARonin • RNA Therapeutics Institute, University of Massachusetts Medical School,
Worcester, MA, USA; Department of Medicine, University of Massachusetts Medical
School, Worcester, MA, USA
SAwen BAkR • Cardiovascular Research Center, Massachusetts General Hospital, Boston,
MA, USA
leonoRA BAlAj • Department of Neurosurgery, Massachusetts General Hospital and
Harvard Medical School, Boston, MA, USA
dAvid BAxteR • Institute for Systems Biology, Seattle, WA, USA
vAlentin x. BeRdAh • Department of Transplantion, Mayo Clinic, Jacksonville, FL, USA
dAvid A. BoRRelli • Department of Transplantion, Mayo Clinic, Jacksonville, FL, USA
MARike BRoekMAn • Department of Neurology, Massachusetts General Hospital and
Harvard Medical School, Boston, MA, USA; Department of Neurosurgery, Brain Center
Rudolf Magnus University Medical Center Utrecht, Utrecht, The Netherlands
AlfonSo CAyotA • Functional Genomics Unit, Institut Pasteur de Montevideo,
Montevideo, Uruguay; Department of Medicine, Faculty of Medicine, Universidad de la
República, Montevideo, Uruguay
liliAn CRuz • Department of Neurology, Massachusetts General Hospital and Harvard
Medical School, Boston, MA, USA
kiRSty M. dAnielSon • Cardiovascular Research Center, Massachusetts General Hospital,
Boston, MA, USA
SAuMyA dAS • Cardiovascular Research Center, Massachusetts General Hospital, Boston,
MA, USA
MARie-CeCile didiot • RNA Therapeutics Institute, University of Massachusetts Medical
School, Worcester, MA, USA; Department of Molecular Medicine, University of
Massachusetts Medical School, Worcester, MA, USA
Sophie A. duSoSwA • Department of Neurology, Massachusetts General Hospital and
Harvard Medical School, Boston, MA, USA; Department of Molecular Cell Biology and
Immunology, VU University Medical Center, Amsterdam, The Netherlands; Amsterdam
Infection & Immunity Institute, VU University Medical Center, Amsterdam,
The Netherlands; Cancer Center Amsterdam, VU University Medical Center,
Amsterdam, The Netherlands
Alton etheRidge • Pacific Northwest Research Institute, Seattle, WA, USA
juStynA filAnt • Department of Gynecologic Oncology and Reproductive Medicine, The
University of Texas MD Anderson Cancer Center (MDACC), Houston, TX, USA
dAvid gAlAS • Pacific Northwest Research Institute, Seattle, WA, USA
RoopAli gAndhi • Department of Neurology, Center for Neurologic Diseases, Brigham
and Women’s Hospital, Harvard Medical School, Boston, MA, USA
AleSSAndRA guRtneR • Department of Neurosurgery, Massachusetts General Hospital and
Harvard Medical School, Boston, MA, USA; Department of Life Sciences, University of
Trieste, Trieste, Italy
ix
x Contributors
BenCe gyoRgy • Department of Neurology, Massachusetts General Hospital and Harvard
Medical School, Boston, MA, USA
RekA A. hARASzti • RNA Therapeutics Institute, University of Massachusetts Medical
School, Worcester, MA, USA; Department of Molecular Medicine, University of
Massachusetts Medical School, Worcester, MA, USA
pete heinzelMAn • Department of Neuroscience, Mayo Clinic-Jacksonville, Jacksonville,
FL, USA
king yeung hong • Department of Surgery, University of California & VA Medical
Center, San Francisco, CA, USA
MiChelle e. hung • Interdisciplinary Biological Sciences Program, Northwestern
University, Evanston, IL, USA
AnAStASiA khvoRovA • RNA Therapeutics Institute, University of Massachusetts Medical
School, Worcester, MA, USA; Department of Molecular Medicine, University of
Massachusetts Medical School, Worcester, MA, USA
Roy y. kiM • Department of Surgery, University of California & VA Medical Center,
San Francisco, CA, USA
louiSe C. lAuRent • Department of Reproductive Medicine, University of California,
San Diego, La Jolla, CA, USA
Stephen B. lenzini • Department of Chemical and Biological Engineering, Northwestern
University, Evanston, IL, USA
joShuA n. leonARd • Department of Chemical and Biological Engineering, Northwestern
University, Evanston, IL, USA; Center for Synthetic Biology, Northwestern University,
Evanston, IL, USA; Chemistry of Life Processes Institute, Northwestern University,
Evanston, IL, USA; Member, Robert H . Lurie Comprehensive Cancer Center,
Northwestern University, Evanston, IL, USA
kAng li • Department of Surgery, University of California & VA Medical Center, San
Francisco, CA, USA
RiShABh lohRAy • Department of Transplantation, Mayo Clinic, Jacksonville, FL, USA
fu SAng luk • Department of Surgery, University of California & VA Medical Center,
San Francisco, CA, USA
Akiko MAtSudA • Department of Transplantation, Mayo Clinic, Jacksonville, FL, USA;
Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA
pARhAM nejAd • Department of Neurology, Center for Neurologic Diseases, Brigham and
Women’s Hospital, Harvard Medical School, Boston, MA, USA
tuShAR pAtel • Department of Transplantion, Mayo Clinic, Jacksonville, FL, USA;
Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA
Anu pAul • Department of Neurology, Center for Neurologic Diseases, Brigham and
Women’s Hospital, Harvard Medical School, Boston, MA, USA
RoBeRt l. RAffAi • Department of Surgery, University of California & VA Medical
Center, San Francisco, CA, USA
RodoStheniS S. RodoSthenouS • Cardiovascular Division of the Massachusetts General
Hospital and Harvard Medical School, Boston, MA, USA
noAh SAdik • Department of Neurosurgery, Massachusetts General Hospital and Harvard
Medical School, Boston, MA, USA
Ane MiRen SAlvAdoR-gARiCAno • Cardiovascular Division of the Massachusetts General
Hospital and Harvard Medical School, Boston, MA, USA