Basic Science Methods for Clinical Researchers

357 Pages·2017·20.954 MB·English

by  Morteza Jalali, Francesca Yvonne Louise Saldanha and Mehdi Jalali (Eds.)

Basic Science Methods
for Clinical Researchers
Basic Science Methods
for Clinical Researchers
Edited by
Morteza Jalali MB ChB PhD (Cantab) MRes BSc
MRCS (Eng) FCPS
University of Oxford, Oxford, United Kingdom
Francesca Y.L. Saldanha MB BChir MA (Cantab)
Whitehead Institute, Massachusetts Institute of Technology, Cambridge,
MA, United States
Mehdi Jalali MB ChB (Hons) MPhil
University of Liverpool, Liverpool, United Kingdom
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Notices
Knowledge and best practice in this field are constantly changing. As new research and
experience broaden our understanding, changes in research methods, professional practices,
or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge
in evaluating and using any information, methods, compounds, or experiments described
herein. In using such information or methods they should be mindful of their own safety and
the safety of others, including parties for whom they have a professional responsibility.
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ISBN: 978-0-12-803077-6
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Dedication
To all current clinician scientists in training, without
whom this book would never have been inspired.
To our unwaveringly supportive families,
without whom this book would not
have been possible.
To you, the reader, may this book guide, excite, and
encourage your pursuit of tomorrow’s advancements in
healthcare that stem from basic scientific research.
List of Contributors
Enrique Amaya University of Manchester, Manchester, United Kingdom
Imad M. Awan Ragon Institute of MGH, MIT and Harvard, Cambridge, MA,
United States
Hugo J. Bellen Department of Molecular and Human Genetics, Baylor College of
Medicine (BCM), Houston, TX, United States; Jan and Dan Duncan Neurological
Research Institute, Texas Children’s Hospital, Houston, TX, United States;
Howard Hughes Medical Institute, Houston, TX, United States
Alessandro Bertero University of Cambridge, Cambridge, United Kingdom
David Brindley University of Oxford, Oxford, United Kingdom; University College
London, London, United Kingdom; Harvard Stem Cell Institute, Cambridge, MA,
United States; USCF-Stanford Center of Excellence in Regulatory Science and
Innovation (CERSI), San Fransisco, CA, United States
Stephanie Brown University of Cambridge, Cambridge, United Kingdom
Eleanor Jane Budge University of Oxford, Oxford, United Kingdom
Eleni Chantzoura University of Edinburgh, Edinburgh, United Kingdom
John Collin University of Bristol Medical School, Bristol, United Kingdom
Amanda S. Coutts University of Oxford, Oxford, United Kingdom
Karel Dorey University of Manchester, Manchester, United Kingdom
Jefte M. Drijvers Ragon Institute of MGH, MIT and Harvard, Cambridge, MA,
United States
Tomasz Gwozdz University of Manchester, Manchester, United Kingdom
Gregory A. Hawkins Wake Forest School of Medicine, Winston-Salem, NC,
United States
Shoko Ishibashi University of Manchester, Manchester, United Kingdom
Tyler Jacks David H. Koch Institute for Integrative Cancer Research, Massachusetts
Institute of Technology, Cambridge, MA, United States; Howard Hughes Medical
Institute, Massachusetts Institute of Technology, Cambridge, MA, United States
Mehdi Jalali University of Liverpool, Liverpool, United Kingdom
Morteza Jalali University of Oxford, Oxford, United Kingdom
Sonali Joshi University of Texas MD Anderson Cancer Center, Houston, TX,
United States
Keisuke Kaji University of Edinburgh, Edinburgh, United Kingdom
xix
xx List of Contributors
Nicholas B. La Thangue University of Oxford, Oxford, United Kingdom
Laurens J. Lambert David H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA, United States
Robert Lea University of Manchester, Manchester, United Kingdom
Martyna Lukoseviciute University of Manchester, Manchester, United Kingdom
Paul Martin University of Bristol Medical School, Bristol, United Kingdom
Mandar D. Muzumdar David H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA, United States; Dana-
Farber Cancer Institute, Boston, MA, United States; Harvard Medical School,
Boston, MA, United States
Frederick D. Park University of California San Diego School of Medicine, La Jolla,
CA, United States; Sanford Consortium for Regenerative Medicine, La Jolla, CA,
United States
Cory A. Perugino Ragon Institute of MGH, MIT and Harvard, Cambridge, MA,
United States
Adam Pettitt Royal Veterinary College, London, United Kingdom
David Pettitt University of Oxford, Oxford, United Kingdom
Shiv Pillai Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States
Tannishtha Reya University of California San Diego School of Medicine, La Jolla,
CA, United States; Sanford Consortium for Regenerative Medicine, La Jolla, CA,
United States
William M. Rideout III David H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA, United States
MacKenna Roberts University of Oxford, Oxford, United Kingdom
Ian M. Rosenberg Ragon Institute of MGH, MIT and Harvard, Cambridge, MA,
United States
Francesca Y.L. Saldanha Massachusetts Institute of Technology, MA, United States
Roman Sasik University of California San Diego School of Medicine, La Jolla, CA,
United States
Charis-P. Segeritz University of Cambridge, Cambridge, United Kingdom
Semira Sheikh University of Oxford, Oxford, United Kingdom
James Smith University of Oxford, Oxford, United Kingdom
Ludovic Vallier University of Cambridge, Cambridge, United Kingdom; Wellcome
Trust Sanger Institute, Hinxton, United Kingdom
Michael F. Wangler Department of Molecular and Human Genetics, Baylor College of
Medicine (BCM), Houston, TX, United States; Jan and Dan Duncan Neurological
Research Institute, Texas Children’s Hospital, Houston, TX, United States
Dihua Yu University of Texas MD Anderson Cancer Center, Houston, TX,
United States
Justyna Zaborowska University of Oxford, Oxford, United Kingdom
Foreword
Professor Dame Sally C. Davies FRS FMedSci
Chief Medical Officer, UK Department of Health, London, England
This first edition of Basic Science Methods for Clinical Researchers represents
a unique approach for the training of clinicians who endeavor to translate basic
scientific research into real healthcare benefits for patients.
There is now international recognition of the need for clinicians who are
dually trained in medicine and science, and thus exceptionally equipped to har-
ness the translational potential of laboratory discoveries and permit their safe
delivery to the bedside. This welcomed and timely volume comes as we cel-
ebrate a decade since our National Institute for Health Research (NIHR) was
established here in the United Kingdom, an organization which sets out to drive
research from bench to bedside by supporting the most talented investigators.
Basic Science Methods for Clinical Researchers will support the endeavors of
such investigators and those aspiring to be, enjoying expert contributions from
research laboratories at the world’s foremost universities.
Accessible and concise in its format, this book will be embraced as a trusted
bench and home study companion by clinicians and medical students globally,
helping to facilitate the development of tomorrow’s leaders in discovery sci-
ence. I applaud the Editors on achieving their goal of putting together the best
possible resource for those embarking on laboratory-based research and wish
all those who are pursuing a career in translational science great success.
xxi
Preface
Biomedical research drives healthcare innovation in the prevention, diagnosis,
and treatment of a wide array of illnesses. There is growing recognition of the
need for clinicians who are scientifically trained (clinician scientists) to take
laboratory discoveries from bench to bedside, illuminating new therapies for
patients. The global move toward recruiting such a cohort of individuals, dually
trained in medicine and science, presents an interesting paradox. There is an
expectation that by the end of medical school, clinicians are well-versed in cel-
lular and molecular biology and related experimental techniques. However,
increasingly pressurized undergraduate medical school curricula now lean
toward early patient exposure and clinical case-based learning, resulting in an
ever-diminishing emphasis on basic science. This is later compounded by a lack
of exposure to basic science due to the heavy time commitment demanded by
clinical work. Thus the aspiring contemporary clinician scientist faces difficult
challenges in learning to simultaneously navigate both the medical and scien-
tific worlds. The onus is now, more than ever, on clinicians to independently
pursue their interest in basic science and proactively seek research training
opportunities outside of formalized clinical training. With the foundations of
basic science and good laboratory practice no longer being provided in medical
school, there is an inevitably steep learning curve when transitioning from the
clinical to the laboratory environment. It can leave many feeling overwhelmed
and without the necessary skills required to address scientific research questions
experimentally.
Basic Science Methods for Clinical Researchers addresses the specific chal-
lenges faced by clinicians without a conventional science background. It is the first
book of its kind to support the research endeavors of aspiring clinician scientists,
in an engaging and accessible format. It seeks to ease the “culture shock” of the
medicine to science transition and provide a platform for understanding how core
experimental methods can be applied in biomedical research. We hope that Basic
Science Methods for Clinical Researchers will be a vital companion for health-
care professionals globally, who are undertaking laboratory-based basic scientific
research. It aims to support clinicians in the pursuit of their academic interests and
in making an original contribution to their chosen fields. In this way, Basic Science
Methods for Clinical Researchers will facilitate the necessary development of
tomorrow’s clinician scientists and future leaders in translational medicine.
xxiii
About This Book
Basic Science Methods for Clinical Researchers was carefully conceptualized
and designed by Morteza Jalali and his co-editors, who together assembled a
host of world-class contributors from leading higher education institutions to
provide unique and expert perspectives in their scientific fields of interest. All
those involved in this project were inspired by the common frustrations faced
by today’s clinician scientists in training and were united in their pursuit of
creating an invaluable resource for those undertaking laboratory-based science.
Basic Science Methods for Clinical Researchers provides a foundation in
the principles and practice of core experimental techniques commonly used to
answer questions in basic scientific research. The book is organized around four
research themes pertaining to key biological molecules, from genes to proteins
to cells, and model organisms. All the chapters follow a standardized format and
take a uniform approach to describing, explaining, and critiquing the featured
research method. Each includes protocols, techniques for troubleshooting com-
mon problems, and importantly an explanation of the advantages and limitations
of a technique in generating conclusive data. In this way the reader is left with
a powerful understanding of each of the techniques covered. At the end of this
book there are three appendices which provide resources for practical research
methodology including legal frameworks for using stem cells and animals in the
laboratory, ethical considerations, and good laboratory practice (GLP).
Basic Science Methods for Clinical Researchers is unique to the market,
providing enough detail to permit the new and inexperienced researcher to
approach a given research technique with confidence, as well as serving as a
useful refresher and quick reference point for those who are more seasoned in
their research career. Each chapter underwent multiple rounds of editing and
revisions until all contributors were happy with the end result—an engaging
and accessible text that inspires and gives confidence to anyone wishing to
undertake basic science training and produce high-quality research. Those who
have been involved in this project from around the world, sincerely hope that
you enjoy reading Basic Science Methods for Clinical Researchers and wel-
come your feedback for future editions, in order to help maintain this book as
a premier text for aspiring clinician scientists. May Basic Science Methods for
Clinical Researchers be an indispensable companion for this most rewarding
and exciting journey between medicine and science.
xxv
Chapter 1
The Polymerase Chain Reaction:
PCR, qPCR, and RT-PCR
Mehdi Jalali1, Justyna Zaborowska2 and Morteza Jalali2
1University of Liverpool, Liverpool, United Kingdom; 2University of Oxford, Oxford,
United Kingdom
Chapter Outline
Introduction 2 Applications of PCR 14
In Principle 2 Scenario 15
Analysis of the PCR Product 3 Key Limitations 15
Phases of PCR 5 Troubleshooting 15
Variations of PCR Method: Little or No PCR Product 15
Quantitative PCR 5 Efficiency of Reaction Is Too Low 15
Variations of PCR Method: Efficiency of Reaction Is Too High 16
Reverse Transcription PCR 8 Multiple Bands On Gel, Or
In Practice 9 Multiple Peaks On Melting Curve 16
Extraction of Total RNA 9 No RT Control Produces a Band 16
Synthesis of cDNA by Reverse Negative PCR Control Produces
Transcription 11 a Band 16
Real-Time PCR Amplification 11 Conclusion 16
Data Analysis: Generating a Standard References 17
Curve 12 Suggested Further Reading 18
Relative Quantification Glossary 18
(Pfaffl Method) 13 List of Acronyms and Abbreviations 18
Objectives
● Describe the scientific principles behind PCR
● Provide PCR laboratory protocols
● List PCR applications
● Describe a typical scenario in which qRT-PCR might be used
● Discuss key limitations and troubleshooting
Basic Science Methods for Clinical Researchers. DOI:http://dx.doi.org/10.1016/B978-0-12-803077-6.00001-1
© 2017Published by Elsevier Inc. All rights reserved. 1