Gene Expression Analysis Methods and Protocols
Edited by
Nalini Raghavachari
Division of Geriatrics and Clinical Gerontology, National Institute on Aging, Bethesda, MD, USA
Natàlia Garcia-Reyero
Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, MS, USA
Editors
Nalini Raghavachari Division of Geriatrics
and Clinical Gerontology National Institute on Aging Bethesda, MD, USA

Nata`lia Garcia-Reyero
Environmental Laboratory
US Army Engineer Research and Development Center Vicksburg, MS, USA

ISSN 1064-3745
Methods in Molecular Biology
ISBN 978-1-4939-7833-5
https://doi.org/10.1007/978-1-4939-7834-2
ISSN 1940-6029 (electronic) ISBN 978-1-4939-7834-2 (eBook)
Library of Congress Control Number: 2018941461
© Springer Science+Business Media, LLC, part of Springer Nature 2018
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Preface
Rapid advances in genomic technologies and computational tools are revolutionizing the field of transcriptomics, which is the study of the transcriptome or the genes transcribed from genomic DNA. The applications of these techniques are endless. From understanding development and disease to environmental monitoring, or community analysis, the pub- lished manuscripts using gene expression to tackle different questions have not stopped and increasing exponentially over the last decades. The methods and techniques are getting more accurate and the computational tools more precise. We can now understand what happens at the single-cell level, which was unthinkable just a few years ago. It is an exciting era with fast advances and new applications that help us understand life.
This volume of the Methods in Molecular Biology series presents a collection of chapters for both experimental and bioinformatics approaches related to different aspects of gene expression analysis. Each chapter begins with an introduction or background of the protocol or technology being described. When appropriate, the Materials section lists all the reagents and other materials needed. A detailed step-by-step description of the protocol used is provided, with the goal of communicating all the practical steps necessary to successfully perform the protocol. In order to provide more useful information, a Notes section is also supplied, with tips and suggestions to best perform the protocol or overcome problems that might arise. In addition to the regular chapters, we have a few review chapters that talk about the current and future trends in gene expression analyses both in wetlab and data analysis. This particular book addresses protocols and techniques related to gene expression. It is divided in three main sections: background chapters, wetlab protocols, and bioinformatics approaches. We have assembled an exciting array of chapters tackling cutting-edge techni- ques, such as single-cell gene expression, highly multiplexed amplicon sequencing, multi- omics techniques, targeted sequencing, or epigenetics. Gene expression analysis is broadly used worldwide and has an immense array of applications. Furthermore, the techniques and methods used are rapidly changing. They are also becoming increasingly precise, informa- tive, often challenging, and applicable to many different fields. That is why we hope that these detailed chapters will provide useful information to researchers worldwide. We greatly appreciate all the authors’ contributions; without their efforts and expertise this book would not have been possible. We hope that this volume will be a valuable part of many labora- tories, hopefully getting old on the bench rather than staying flawless on the library shelf.
Bethesda, MD, USA Vicksburg, MS, USA
Nalini Raghavachari Nat`alia Garcia-Reyero
v
Contents
Preface..................................................................... v Contributors................................................................. ix
  1. 1  Overview of Gene Expression Analysis: Transcriptomics ..................... 1
    Nalini Raghavachari and Nat`alia Garcia-Reyero
  2. 2  RNA-Seq and Expression Arrays: Selection Guidelines forGenome-WideExpressionProfiling.................................... 7 Jessica Minnier, Nathan D. Pennock, Quichen Guo, Pepper Schedin,
    and Christina A. Harrington

  3. 3  A Guide for Designing and Analyzing RNA-Seq Data ....................... 35
    Aniruddha Chatterjee, Antonio Ahn, Euan J. Rodger, Peter A. Stockwell, and Michael R. Eccles
  4. 4  SureSelectXT RNA Direct: A Technique for Expression Analysis
    Through Sequencing of Target-Enriched FFPE Total RNA .................. 81
    Jennifer Carter Jones, Alex P. Siebold, Carolina Becker Livi,
    and Anne Bergstrom Lucas

  5. 5  Simultaneous, Multiplexed Detection of RNA and Protein
    ontheNanoString
    ® nCounter® Platform.................................. 105 Sarah Warren
  6. 6  Transcript Profiling Using Long-Read Sequencing Technologies . . . . . . . . . . . . . 121
    Anthony Bayega, Yu Chang Wang, Spyros Oikonomopoulos,
    Haig Djambazian, Somayyeh Fahiminiya, and Jiannis Ragoussis

  7. 7  Making and Sequencing Heavily Multiplexed, High-Throughput
    16S Ribosomal RNA Gene Amplicon Libraries Using a Flexible, Two-StagePCRProtocol................................................ 149
    Ankur Naqib, Silvana Poggi, Weihua Wang, Marieta Hyde,
    Kevin Kunstman, and Stefan J. Green

  8. 8  MicroRNA Expression Analysis: Next-Generation Sequencing. . . . . . . . . . . . . . . . 171
    Poching Liu
  9. 9  Identification of Transcriptional Regulators That Bind to Long
    Noncoding RNAs by RNA Pull-Down and RNA Immunoprecipitation . . . . . . . 185
    Xiangbo Ruan, Ping Li, and Haiming Cao
  10. 10  Single-Cell mRNA-Seq Using the Fluidigm C1 System
    and Integrated Fluidics Circuits .......................................... 193
    Haibiao Gong, Devin Do, and Ramesh Ramakrishnan
  11. 11  Current and Future Methods for mRNA Analysis: A Drive
    Toward Single Molecule Sequencing ...................................... 209
    Anthony Bayega, Somayyeh Fahiminiya, Spyros Oikonomopoulos,
    and Jiannis Ragoussis

vii
viii Contents
  1. 12  Expression Profiling of Differentially Regulated Genes inFanconiAnemia...................................................... 243 Binita Zipporah E, Kavitha Govarthanan, Pavithra Shyamsunder,
    and Rama S. Verma

  2. 13  A Review of Transcriptome Analysis in Pulmonary Vascular Diseases . . . . . . . . . . 259
    Dustin R. Fraidenburg and Roberto F. Machado
  3. 14  Differential Gene Expression Analysis of Plants............................. 279
    Mark Arick II and Chuan-Yu Hsu
  4. 15  High Throughput Sequencing-Based Approaches for Gene
    Expression Analysis ..................................................... 299
    R. Raja Sekhara Reddy and M. V. Ramanujam
  5. 16  Network Analysis of Gene Expression ..................................... 325
    Roby Joehanes
  6. 17  Analysis of ChIP-Seq and RNA-Seq Data with BioWardrobe................. 343
    Sushmitha Vallabh, Andrey V. Kartashov, and Artem Barski
  7. 18  Bayesian Network to Infer Drug-Induced Apoptosis Circuits
    from Connectivity Map Data............................................. 361
    Jiyang Yu and Jose M. Silva
Index...................................................................... 379
Contributors
ANTONIO AHN Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand
MARK ARICK II Institute for Genomics, Biocomputing & Biotechnology, Mississippi State University, Mississippi State, MS, USA
ARTEM BARSKI Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA
ANTHONY BAYEGA Department of Human Genetics, McGill University and Genome Quebec Innovation Centre, McGill University, Montre ́al, QC, Canada
HAIMING CAO Cardiovascular Branch, National Heart, Lung and Blood Institute, NIH, Bethesda, MD, USA
ANIRUDDHA CHATTERJEE Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand
HAIG DJAMBAZIAN Department of Human Genetics, McGill University and Genome Quebec Innovation Centre, McGill University, Montre ́al, QC, Canada
DEVIN DO Fluidigm Corporation, South San Francisco, CA, USA
MICHAEL R. ECCLES Department of Pathology, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery,
Auckland, New Zealand
SOMAYYEH FAHIMINIYA Department of Human Genetics, McGill University and Genome
Quebec Innovation Centre, McGill University, Montre ́al, QC, Canada; Cancer Research Program, The Research Institute of the McGill University Health Centre, Montreal, QC, Canada
DUSTIN R. FRAIDENBURG Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA
NATA` LIA GARCIA-REYERO Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, MS, USA
HAIBIAO GONG Fluidigm Corporation, South San Francisco, CA, USA
KAVITHA GOVARTHANAN Department of Biotechnology, Bhupat and Jyoti Mehta School of
Biosciences, Stem Cell and Molecular Biology Lab, Indian Institute of Technology Madras,
Chennai, India
STEFAN J. GREEN DNA Services Facility, Research Resources Center, University of Illinois at
Chicago, Chicago, IL, USA
QUICHEN GUO Department of Cell, Developmental and Cancer Biology, Oregon Health
and Science University, Portland, OR, USA
CHRISTINA A. HARRINGTON Department of Molecular and Medical Genetics, Integrated
Genomics Laboratory, Oregon Health and Science University, Portland, OR, USA CHUAN-YU HSU Institute for Genomics, Biocomputing & Biotechnology, Mississippi State
University, Mississippi State, MS, USA
MARIETA HYDE DNA Services Facility, Research Resources Center, University of Illinois at
Chicago, Chicago, IL, USA
ix
x Contributors
ROBY JOEHANES Hebrew SeniorLife, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
JENNIFER CARTER JONES Agilent Technologies, Santa Clara, CA, USA
ANDREY V. KARTASHOV Division of Allergy and Immunology, Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH, USA
KEVIN KUNSTMAN DNA Services Facility, Research Resources Center, University of Illinois
at Chicago, Chicago, IL, USA
PING LI Cardiovascular Branch, National Heart, Lung and Blood Institute, NIH,
Bethesda, MD, USA
POCHING LIU DNA Sequencing and Genomics Core—NHLBI, National Institute
of Health, Bethesda, MD, USA
CAROLINA BECKER LIVI ANNE BERGSTROM LUCAS ROBERTO F. MACHADO
Agilent Technologies, Santa Clara, CA, USA
Agilent Technologies, Santa Clara, CA, USA
Department of Medicine, Division of Pulmonary, Critical Care,

Sleep and Allergy, University of Illinois at Chicago, Chicago, IL, USA; Department
of Medicine, Division of Pulmonary, Critical Care, Sleep, and Occupational Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

JESSICA MINNIER School of Public Health, Oregon Health and Science University, Portland, OR, USA
ANKUR NAQIB DNA Services Facility, Research Resources Center, University of Illinois at Chicago, Chicago, IL, USA
SPYROS OIKONOMOPOULOS Department of Human Genetics, McGill University and Genome Quebec Innovation Centre, McGill University, Montre ́al, QC, Canada
NATHAN D. PENNOCK Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University, Portland, OR, USA
SILVANA POGGI DNA Services Facility, Research Resources Center, University of Illinois at Chicago, Chicago, IL, USA
NALINI RAGHAVACHARI Division of Geriatrics and Clinical Gerontology, National Institute on Aging, Bethesda, MD, USA
JIANNIS RAGOUSSIS Department of Human Genetics, McGill University and Genome Quebec Innovation Centre, McGill University, Montre ́al, QC, Canada; Department of Bioengineering, McGill University, Montre ́al, QC, Canada; Department of Biochemistry, Center of Innovation in Personalized Medicine, Cancer and Mutagen Unit, King Fahd Center for Medical Research, King Abdulaziz University, Jeddah, Saudi Arabia
R. RAJA SEKHARA REDDY Clevergene Biocorp Private Limited, Bangalore, Karnataka, India
RAMESH RAMAKRISHNAN Fluidigm Corporation, South San Francisco, CA, USA; Dovetail Genomics LLC, Santa Cruz, CA, USA
Clevergene Biocorp Private Limited, Bangalore, Karnataka, India
M. V. RAMANUJAM
EUAN J. RODGER Department of Pathology, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery,
Auckland, New Zealand
XIANGBO RUAN Cardiovascular Branch, National Heart, Lung and Blood Institute, NIH,
Bethesda, MD, USA
PEPPER SCHEDIN Department of Cell, Developmental and Cancer Biology, Knight Cancer
Institute, Oregon Health and Science University, Portland, OR, USA; Young Women’s Breast Cancer Translational Program, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

PAVITHRA SHYAMSUNDER Cancer Science Institute, NUS, Singapore, Singapore ALEX P. SIEBOLD Agilent Technologies, Santa Clara, CA, USA
JOSE M. SILVA Department of Pathology, Icahn School of Medicine at Mount Sinai,
The Mount Sinai Hospital, New York, NY, USA
PETER A. STOCKWELL Department of Biochemistry, University of Otago, Dunedin, New
Zealand
SUSHMITHA VALLABH Division of Allergy and Immunology, Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH, USA
RAMA S. VERMA Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences,
Stem Cell and Molecular Biology Lab, Indian Institute of Technology Madras, Chennai,
India
WEIHUA WANG DNA Services Facility, Research Resources Center, University of Illinois
at Chicago, Chicago, IL, USA
YU CHANG WANG Department of Human Genetics, McGill University and Genome
Quebec Innovation Centre, McGill University, Montre ́al, QC, Canada
SARAH WARREN NanoString Technologies, Seattle, WA, USA
JIYANG YU Department of Computational Biology, St. Jude Children’s Research Hospital,
Memphis, TN, USA
BINITA ZIPPORAH E Department of Biotechnology, Bhupat and Jyoti Mehta School of
Biosciences, Stem Cell and Molecular Biology Lab, Indian Institute of Technology Madras, Chennai, India 

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