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Methods in
Molecular Biology 2588

Gregory J. Seymour
Mary P. Cullinan
Nicholas C.K. Heng
Paul R. Cooper Editors

Oral Biology
Molecular Techniques
and Applications
Third Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

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For over 35 years, biological scientists have come to rely on the research protocols and
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 Oral Biology

Molecular Techniques and Applications

Third Edition

Edited by

Gregory J. Seymour and Mary P. Cullinan

School of Dentistry, The University of Queensland, Herston, QLD, Australia

Nicholas C. K. Heng and Paul R. Cooper

Faculty of Dentistry, University of Otago, Dunedin, New Zealand
Editors
Gregory J. Seymour Mary P. Cullinan
School of Dentistry School of Dentistry
The University of Queensland The University of Queensland
Herston, QLD, Australia Herston, QLD, Australia

Nicholas C. K. Heng Paul R. Cooper

Faculty of Dentistry Faculty of Dentistry
University of Otago University of Otago
Dunedin, New Zealand Dunedin, New Zealand

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
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Preface

The biological and physical sciences remain the research base that underpins advances in the
prevention and treatment of oral diseases, and it is in this context that this third edition
continues the theme of the very successful previous editions of Oral Biology: Molecular
Techniques and Applications. As in previous editions, it is recognized that it is not possible to
include all possible techniques in a single volume. Nevertheless, many of the techniques
covered in the previous editions remain valid and have been extensively updated and
included in this edition together with 14 new chapters. In recent years, there have been
major advances in the field of regenerative biology such that two of the new chapters cover
3D printing and cell seeding of 3D scaffolds. Chapters on gene editing and the use of
CRISPR in oral biology, and histone acetylation and deacetylation techniques are also in this
edition, further reflecting advances in the application of molecular techniques to oral
biology. In presenting a selection of up-to-date molecular methods applicable to the study
of oral health and disease, it is hoped that this third edition of Oral Biology: Molecular
Techniques and Applications will continue to be a basic resource not only for new researchers
but also for experienced scientists wishing to expand their research platform into new areas
of oral biology.

Herston, QLD, Australia Gregory J. Seymour

Mary P. Cullinan
Dunedin, New Zealand Nicholas C. K. Heng
Paul R. Cooper

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I SALIVA AND OTHER ORAL FLUIDS

1 RNA Sequencing Analysis of Saliva exRNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Karolina Elżbieta Kaczor-Urbanowicz and David T. W. Wong
2 Proteome Analysis of Oral Biofluids in Periodontal Health and Disease
Using Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Nagihan Bostanci and Kai Bao
3 Saliva Diagnosis Using Small Extracellular Vesicles and Salivaomics . . . . . . . . . . . 25
Pingping Han, Xiang Li, Wei Wei, and Sašo Ivanovski
4 Antioxidant Micronutrients and Oxidative Stress Biomarkers . . . . . . . . . . . . . . . . . 41
Irundika H. K. Dias, Helen R. Griffiths, Mike R. Milward,
Martin R. Ling, Iain L. C. Chapple, and Melissa M. Grant

PART II MOLECULAR BIOSCIENCES

5 The Oral Microbiota in Health and Disease: An Overview

of Molecular Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
José F. Siqueira Jr. and Isabela N. Rôças
6 The Long and Short of Genome Sequencing: Using a Hybrid Sequencing
Strategy to Sequence Oral Microbial Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Nicholas C. K. Heng and Jo-Ann L. Stanton
7 Microbial Community Profiling Using Terminal Restriction Fragment
Length Polymorphism (T-RFLP) and Denaturing Gradient
Gel Electrophoresis (DGGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
José F. Siqueira Jr, Mitsuo Sakamoto, and Alexandre S. Rosado
8 Bioinformatic Approaches for Describing the Oral Microbiota. . . . . . . . . . . . . . . . 105
Kristi Biswas, Michael W. Taylor, and David T. J. Broderick
9 Adhesion of Yeast and Bacteria to Oral Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Richard D. Cannon, Karl M. Lyons, Kenneth Chong,
Kathryn Newsham-West, Kyoko Niimi, and Ann R. Holmes
10 Quantitative Analysis of Periodontal Pathogens Using Real-Time
Polymerase Chain Reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Mª. José Marin, Elena Figuero, David Herrera, and Mariano Sanz
11 Methods to Study Antagonistic Activities Among Oral Bacteria. . . . . . . . . . . . . . . 171
Fengxia Qi and Jens Kreth
12 Generation of Multispecies Oral Bacteria Biofilm Models . . . . . . . . . . . . . . . . . . . . 187
Jason L. Brown, Mark C. Butcher, Chandra Lekha Ramalingam Veena,
Safa Chogule, William Johnston, and Gordon Ramage

vii
viii Contents

13 Markerless Genome Editing in Competent Streptococci . . . . . . . . . . . . . . . . . . . . . 201

Roger Junges, Rabia Khan, Yanina Tovpeko, Heidi A. Åmdal,
Fernanda C. Petersen, and Donald A. Morrison
14 A Protocol to Produce Genetically Edited Primary Oral Keratinocytes
Using the CRISPR-Cas9 System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Sven E. Niklander and Keith D. Hunter
15 Size-Based Method for Enrichment of Circulating Tumor Cells
from Blood of Colorectal Cancer Patients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Sai Shyam Vasantharajan, Edward Barnett, Elin S. Gray,
Euan J. Rodger, Michael R. Eccles, Sharon Pattison,
Fran Munro, and Aniruddha Chatterjee
16 Strategy for RNA-Seq Experimental Design and Data Analysis . . . . . . . . . . . . . . . 249
Gregory Gimenez, Peter A. Stockwell, Euan J. Rodger,
and Aniruddha Chatterjee
17 Characterization of the Expression and Role of Histone Acetylation
and Deacetylation in Dental Pulp Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Yukako Yamauchi and Henry F. Duncan
18 Genome-Wide Analysis of Periodontal and Peri-implant
Cells and Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Moritz Kebschull, Annika Therese Kroeger,
and Panos N. Papapanou
19 Differential Expression, Functional and Machine Learning Analysis
of High-Throughput –Omics Data Using Open-Source Tools. . . . . . . . . . . . . . . . 317
Moritz Kebschull, Annika Therese Kroeger,
and Panos N. Papapanou
20 Micro-RNA Profiling in Dental Pulp Cell Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . 353
Michaela Kearney and Henry F. Duncan

PART III CELLS AND TISSUES

21 Oral Epithelial Cell Culture Model for Studying the Pathogenesis
of Chronic Inflammatory Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Mike R. Milward, Martin R. Ling, Melissa M. Grant,
Joanna Batt, and Iain L. C. Chapple
22 A Cell Culture Method for the Isolation and Study of Primary Human
Dental Pulp Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Shelly Arora, Benedict Seo, Lara Friedlander,
and Haizal Mohd Hussaini
23 Culturing Adipose-Derived Stem Cells Under Serum-Free Conditions . . . . . . . . 407
Diogo Godoy Zanicotti, Trudy J. Milne, and Dawn E. Coates
24 Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts
Using a One-Step System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Dawn E. Coates, Sobia Zafar, and Trudy J. Milne
 Contents ix

25 Fabrication and Characterization of Decellularized Periodontal Ligament

Cell Sheet Constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Amro Farag, Cedryck Vaquette, Dietmar W. Hutmacher,
P. Mark Bartold, and Sašo Ivanovski
26 Immunohistochemistry and Immunofluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Haizal Mohd Hussaini, Benedict Seo, and Alison M. Rich
27 Characterization, Quantification, and Visualization of Neutrophil
Extracellular Traps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Josefine Hirschfeld, Ilaria J. Chicca, Carolyn G. J. Moonen,
Phillipa C. White, Martin R. Ling, Helen J. Wright,
Paul R. Cooper, Mike R. Milward, and Iain L. C. Chapple
28 Cell Seeding on 3D Scaffolds for Tissue Engineering and Disease
Modeling Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
Fanny Blaudez, Cedryck Vaquette, and Sašo Ivanovski
29 Workflow for Fabricating 3D-Printed Resorbable Personalized Porous
Scaffolds for Orofacial Bone Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
Cedryck Vaquette, Danilo Carluccio, Martin Batstone,
and Sašo Ivanovski
30 Methacrylated Gelatin as an On-Demand Injectable Vehicle
for Drug Delivery in Dentistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
W. Benton Swanson, Abdel Hameed Mahmoud, Seth Woodbury,
and Marco C. Bottino
31 In Vitro Biological Testing of Dental Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
Jithendra Ratnayake, Josette Camilleri,
T. Nethmini Haththotuwa, and Jeffrey Huang

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
Contributors

HEIDI A. ÅMDAL • Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo,
Norway
SHELLY ARORA • Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago,
Dunedin, New Zealand
KAI BAO • Section of Oral Health and Periodontology, Division of Oral Diseases, Department
of Dental Medicine, Karolinska Institutet, Stockholm, Sweden
EDWARD BARNETT • Department of Pathology, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand
P. MARK BARTOLD • Colgate Australian Clinical Dental Research Centre, Dental School,
University of Adelaide, Adelaide, Australia
MARTIN BATSTONE • Herston Biofabrication Institute, Metro North Hospital and Health
Service, Brisbane, QLD, Australia
JOANNA BATT • The School of Dentistry, University of Birmingham, Birmingham, UK
KRISTI BISWAS • Department of Surgery, Faculty of Medical and Health Sciences, University
of Auckland, Auckland, New Zealand
FANNY BLAUDEZ • School of Dentistry, The University of Queensland, Brisbane, Australia
NAGIHAN BOSTANCI • Section of Oral Health and Periodontology, Division of Oral Diseases,
Department of Dental Medicine, Karolinska Institutet, Stockholm, Sweden
MARCO C. BOTTINO • Department of Cariology, Restorative Sciences, and Endodontics,
University of Michigan School of Dentistry, Ann Arbor, MI, USA; Department of
Biomedical Engineering, College of Engineering, University of Michigan, Ann Arbor, MI,
USA
DAVID T. J. BRODERICK • Department of Surgery, Faculty of Medical and Health Sciences,
University of Auckland, Auckland, New Zealand; School of Biological Sciences, University
of Auckland, Auckland, New Zealand
JASON L. BROWN • Oral Sciences Research Group, College of Medical, Veterinary and Life
Sciences, Glasgow University, Glasgow, UK; Glasgow Biofilm Research Network, Glasgow,
UK
MARK C. BUTCHER • Oral Sciences Research Group, College of Medical, Veterinary and Life
Sciences, Glasgow University, Glasgow, UK; Glasgow Biofilm Research Network, Glasgow,
UK
JOSETTE CAMILLERI • School of Dentistry, Institute of Clinical Sciences, College of Dental and
Medical Sciences, University of Birmingham, Birmingham, UK
RICHARD D. CANNON • Sir John Walsh Research Institute, Faculty of Dentistry, University of
Otago, Dunedin, New Zealand
DANILO CARLUCCIO • Herston Biofabrication Institute, Metro North Hospital and Health
Service, Brisbane, QLD, Australia
IAIN L. C. CHAPPLE • The School of Dentistry, University of Birmingham, Birmingham, UK;
Department of Periodontology, Birmingham Dental School and Hospital, University of
Birmingham, Birmingham, UK
ANIRUDDHA CHATTERJEE • Department of Pathology, Dunedin School of Medicine, University
of Otago, Dunedin, New Zealand; School of Health Sciences, UPES University, Dehradun,
India

xi
xii Contributors

ILARIA J. CHICCA • Clinical Immunology Service, Medical School, University of Birmingham,

Birmingham, UK
SAFA CHOGULE • Oral Sciences Research Group, College of Medical, Veterinary and Life
Sciences, Glasgow University, Glasgow, UK; Glasgow Biofilm Research Network, Glasgow,
UK
KENNETH CHONG • Sir John Walsh Research Institute, Faculty of Dentistry, University of
Otago, Dunedin, New Zealand
DAWN E. COATES • Sir John Walsh Research Institute, Faculty of Dentistry, University of
Otago, Dunedin, New Zealand
PAUL R. COOPER • Faculty of Dentistry, University of Otago, Dunedin, New Zealand
IRUNDIKA H. K. DIAS • Aston Medical School, Aston University, Birmingham, UK
HENRY F. DUNCAN • Division of Restorative Dentistry and Periodontology, Dublin Dental
University Hospital, Trinity College Dublin, Dublin, Ireland
MICHAEL R. ECCLES • Department of Pathology, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand
AMRO FARAG • School of Dentistry, The University of Queensland, Brisbane, Australia
ELENA FIGUERO • Oral Research Laboratory, Faculty of Dentistry, University Complutense,
Madrid, Spain; Etiology and Therapy of Periodontal and Peri-implant Diseases (ETEP)
Research Group, University Complutense, Madrid, Spain
LARA FRIEDLANDER • Sir John Walsh Research Institute, Faculty of Dentistry, University of
Otago, Dunedin, New Zealand
GREGORY GIMENEZ • Department of Pathology, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand
MELISSA M. GRANT • The School of Dentistry, University of Birmingham, Birmingham, UK
ELIN S. GRAY • Centre for Precision Health, Edith Cowan University, Joondalup, Australia
HELEN R. GRIFFITHS • Faculty of Health & Medical Sciences, University of Surrey, Surrey,
UK
PINGPING HAN • School of Dentistry, The University of Queensland, Brisbane, Australia;
School of Dentistry, Centre for Orofacial Regeneration, Reconstruction and Rehabilitation
(COR3), The University of Queensland, Brisbane, Australia
T. NETHMINI HATHTHOTUWA • Department of Anatomy, School of Biomedical Sciences,
University of Otago, Otago, New Zealand
NICHOLAS C. K. HENG • Sir John Walsh Research Institute, Faculty of Dentistry, University
of Otago, Dunedin, New Zealand
DAVID HERRERA • Etiology and Therapy of Periodontal and Peri-implant Diseases (ETEP)
Research Group, University Complutense, Madrid, Spain
JOSEFINE HIRSCHFELD • Department of Periodontology, Birmingham Dental School and
Hospital, University of Birmingham, Birmingham, UK
ANN R. HOLMES • Sir John Walsh Research Institute, Faculty of Dentistry, University of
Otago, Dunedin, New Zealand
JEFFREY HUANG • Department of Anatomy, School of Biomedical Sciences, University of
Otago, Otago, New Zealand
KEITH D. HUNTER • Unit of Oral and Maxillofacial Medicine and Pathology, School of
Clinical Dentistry, University of Sheffield, Sheffield, UK; Oral Biology and Pathology,
University of Pretoria, Pretoria, South Africa; Liverpool Head and Neck Centre, University
of Liverpool, Liverpool, UK
 Contributors xiii

HAIZAL MOHD HUSSAINI • Sir John Walsh Research Institute, Faculty of Dentistry, University
of Otago, Dunedin, New Zealand; School of Dentistry, University of Otago, Dunedin, New
Zealand
DIETMAR W. HUTMACHER • Centre for Regenerative Medicine, Institute of Health and
Biomedical Innovation, Queensland University of Technology, Brisbane, Australia
SAŠO IVANOVSKI • School of Dentistry, The University of Queensland, Brisbane, Australia;
School of Dentistry, Centre for Orofacial Regeneration, Reconstruction and Rehabilitation
(COR3), The University of Queensland, Brisbane, Australia; Herston Biofabrication
Institute, Metro North Hospital and Health Service, Brisbane, Australia
WILLIAM JOHNSTON • Oral Sciences Research Group, College of Medical, Veterinary and Life
Sciences, Glasgow University, Glasgow, UK; Glasgow Biofilm Research Network, Glasgow,
UK
ROGER JUNGES • Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo,
Norway
KAROLINA ELŻBIETA KACZOR-URBANOWICZ • Center for Oral and Head/Neck Oncology
Research, UCLA School of Dentistry, University of California at Los Angeles, Los Angeles,
CA, USA; UCLA Institute for Quantitative and Computational Biosciences, University of
California at Los Angeles, Los Angeles, CA, USA; UCLA Section of Orthodontics,
University of California at Los Angeles, Los Angeles, CA, USA; Section of Biosystems and
Function, UCLA School of Dentistry, University of California at Los Angeles, Los Angeles,
CA, USA
MICHAELA KEARNEY • Division of Restorative Dentistry and Periodontology, Dublin Dental
University Hospital, Trinity College Dublin, Dublin, Ireland
MORITZ KEBSCHULL • Periodontal Research Group, Institute of Clinical Sciences, College of
Medical & Dental Sciences, The University of Birmingham, Birmingham, UK; Division of
Periodontics, Section of Oral, Diagnostic and Rehabilitation Sciences, Columbia University
College of Dental Medicine, New York, NY, USA; Birmingham Community Healthcare
NHS Trust, Birmingham, UK
RABIA KHAN • Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo,
Norway
JENS KRETH • School of Dentistry, Oregon Health & Science University, Portland, OR, USA
ANNIKA THERESE KROEGER • Birmingham Community Healthcare NHS Trust,
Birmingham, UK; Department of Oral Surgery, School of Dentistry, University of
Birmingham, Birmingham, UK
XIANG LI • Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Wuhan,
China; Medical Research Institute, Wuhan University, Wuhan, China; Department of
Neurosurgery and Brain Research Center, Zhongnan Hospital, Wuhan University,
Wuhan, China
MARTIN R. LING • Oral Health R&D, GSK, Weybridge, UK; Birmingham Dental School
and Hospital, University of Birmingham, Birmingham, UK
KARL M. LYONS • Department of Oral Rehabilitation, University of Otago, Dunedin, New
Zealand
ABDEL HAMEED MAHMOUD • Department of Cariology, Restorative Sciences, and
Endodontics, University of Michigan School of Dentistry, Ann Arbor, MI, USA
Mª. JOSÉ MARIN • Oral Research Laboratory, Faculty of Dentistry, University Complutense,
Madrid, Spain; Etiology and Therapy of Periodontal and Peri-implant Diseases (ETEP)
Research Group, University Complutense, Madrid, Spain
xiv Contributors

TRUDY J. MILNE • Sir John Walsh Research Institute, Faculty of Dentistry, University of
Otago, Dunedin, New Zealand
MIKE R. MILWARD • The School of Dentistry, University of Birmingham, Birmingham, UK;
Department of Periodontology, Birmingham Dental School and Hospital, University of
Birmingham, Birmingham, UK
CAROLYN G. J. MOONEN • Bruker Corporation, Leiderdorp, the Netherlands; Academic
Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije
Universiteit Amsterdam, Amsterdam, the Netherlands
DONALD A. MORRISON • Department of Biological Sciences, College of Liberal Arts and
Sciences, University of Illinois at Chicago, Chicago, IL, USA
FRAN MUNRO • Department of Surgical Sciences, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand
KATHRYN NEWSHAM-WEST • Department of Oral Rehabilitation, University of Otago,
Dunedin, New Zealand
KYOKO NIIMI • Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago,
Dunedin, New Zealand
SVEN E. NIKLANDER • Unidad de Patologı́a y Medicina Oral, Facultad de Odontologia,
Universidad Andres Bello, Viña del Mar, Chile
PANOS N. PAPAPANOU • Division of Periodontics, Section of Oral, Diagnostic and
Rehabilitation Sciences, Columbia University College of Dental Medicine, New York, NY,
USA
SHARON PATTISON • Department of Medicine, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand
FERNANDA C. PETERSEN • Department of Oral Biology, Faculty of Dentistry, University of
Oslo, Oslo, Norway
FENGXIA QI • University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
GORDON RAMAGE • Oral Sciences Research Group, College of Medical, Veterinary and Life
Sciences, Glasgow University, Glasgow, UK; Glasgow Biofilm Research Network, Glasgow,
UK
JITHENDRA RATNAYAKE • Faculty of Dentistry, Department of Oral Sciences, University of
Otago, Dunedin, New Zealand
ALISON M. RICH • School of Dentistry, University of Otago, Dunedin, New Zealand; School of
Medicine, University of Otago, Dunedin, New Zealand
ISABELA N. RÔÇAS • Department of Endodontics and Molecular Microbiology, Iguaçu
University, Rio de Janeiro, Brazil
EUAN J. RODGER • Department of Pathology, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand
ALEXANDRE S. ROSADO • Institute of Microbiology Prof. Paulo de Goes, Federal University of
Rio de Janeiro, Rio de Janeiro, Brazil
MITSUO SAKAMOTO • Microbe Division/Japan Collection of Microorganisms, RIKEN
BioResource Center, Saitama, Japan
MARIANO SANZ • Etiology and Therapy of Periodontal and Peri-implant Diseases (ETEP)
Research Group, University Complutense, Madrid, Spain
BENEDICT SEO • Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago,
Dunedin, New Zealand; School of Dentistry, University of Otago, Dunedin, New Zealand
JOSÉ F. SIQUEIRA JR • Department of Endodontics and Molecular Microbiology, Iguaçu
University, Rio de Janeiro, Brazil
 Contributors xv

JO-ANN L. STANTON • Department of Anatomy, University of Otago, Dunedin, New

Zealand
PETER A. STOCKWELL • Department of Pathology, Dunedin School of Medicine, University of
Otago, Dunedin, New Zealand
W. BENTON SWANSON • Department of Biologic and Materials Science and Division of
Prosthodontics, University of Michigan School of Dentistry, Ann Arbor, MI, USA
MICHAEL W. TAYLOR • School of Biological Sciences, University of Auckland, Auckland, New
Zealand
YANINA TOVPEKO • Department of Biological Sciences, College of Liberal Arts and Sciences,
University of Illinois at Chicago, Chicago, IL, USA
CEDRYCK VAQUETTE • School of Dentistry, The University of Queensland, Brisbane, Australia;
School of Dentistry, Centre for Orofacial Regeneration, Reconstruction and Rehabilitation
(COR3), The University of Queensland, Brisbane, Australia; Herston Biofabrication
Institute, Metro North Hospital and Health Service, Brisbane, Australia
SAI SHYAM VASANTHARAJAN • Department of Pathology, Dunedin School of Medicine,
University of Otago, Dunedin, New Zealand
CHANDRA LEKHA RAMALINGAM VEENA • Oral Sciences Research Group, College of Medical,
Veterinary and Life Sciences, Glasgow University, Glasgow, UK; Glasgow Biofilm Research
Network, Glasgow, UK
WEI WEI • Department of Neurosurgery, Zhongnan Hospital, Wuhan University, Wuhan,
China; Department of Neurosurgery and Brain Research Center, Zhongnan Hospital,
Wuhan University, Wuhan, China
PHILLIPA C. WHITE • Birmingham Dental School and Hospital, University of Birmingham,
Birmingham, UK
DAVID T. W. WONG • Center for Oral and Head/Neck Oncology Research, UCLA School of
Dentistry, University of California at Los Angeles, Los Angeles, CA, USA; Section of
Biosystems and Function, UCLA School of Dentistry, University of California at Los
Angeles, Los Angeles, CA, USA; UCLA’s Jonsson Comprehensive Cancer Center, Los
Angeles, CA, USA
SETH WOODBURY • Department of Biologic and Materials Science and Division of
Prosthodontics, University of Michigan School of Dentistry, Ann Arbor, MI, USA
HELEN J. WRIGHT • Birmingham Dental School and Hospital, University of Birmingham,
Birmingham, UK
YUKAKO YAMAUCHI • Division of Restorative Dentistry and Periodontology, Dublin Dental
University Hospital, Trinity College Dublin, Dublin, Ireland
SOBIA ZAFAR • Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago,
Dunedin, New Zealand
DIOGO GODOY ZANICOTTI • Sir John Walsh Research Institute, Faculty of Dentistry,
University of Otago, Dunedin, New Zealand
 Part I

Saliva and Other Oral Fluids

 Chapter 1

RNA Sequencing Analysis of Saliva exRNA

Karolina Elżbieta Kaczor-Urbanowicz and David T. W. Wong

Abstract
Next-generation sequencing (NGS) methodologies are rapidly developing. However, RNA Sequencing of
saliva is challenging due to low abundance and integrity of extracellular RNA, as well as large amounts of
bacterial RNAs that may be encountered in saliva. In addition, the literature about human salivary
extracellular RNA is very scarce. Therefore, in our chapter, we present the most appropriate protocols for
saliva collection, pre- and post-processing, including bioinformatic analysis of salivary RNA Sequencing
data. However, the choice of the proper method for RNA extraction, cDNA library preparation, and
computational pipeline can make a significant impact on the final quality of data and their interpretation.

Key words RNA Sequencing, Saliva, Salivary RNAs, Transcriptome, Bioinformatic analysis

1 Introduction

RNA Sequencing (RNA-Seq) is a rapidly developing tool to tran-

scriptome profiling that uses deep-sequencing technologies and is
becoming the major tool in analyzing gene expression [1]. This is a
new high-throughput method for both mapping and quantifying
transcriptomes. It provides more detailed information about the
levels of transcripts and their isoforms than other methods
[2]. Thus, revealing the transcriptome is crucial for investigating
the functional elements of the genome, the molecular constituents
of cells and tissues, and also for understanding development and
disease [2].
RNA-Seq has clear advantages over existing approaches. It can
be used for detection of known and novel complex transcripts as
well as for precise localization of transcription boundaries, to a
single-base resolution [3, 4]. Having very low background signal,
RNA-Seq significantly differs from microarray platforms by allow-
ing unique mapping to the genome of interest [5]. It also enables

Gregory J. Seymour et al. (eds.), Oral Biology: Molecular Techniques and Applications,
Methods in Molecular Biology, vol. 2588, https://doi.org/10.1007/978-1-0716-2780-8_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

3
4 Karolina Elżbieta Kaczor-Urbanowicz and David T. W. Wong

identification of different transcript variants and quantification of

allele-specific expression within an individual [6–8]. Finally, it has
an accurate and large dynamic range of expression levels with high
reproducibility [3, 5].
However, there are also some problems associated with RNA-
Seq technology, specifically with RNA isolation and complemen-
tary DNA (cDNA) library construction as they include several
manipulation stages, thus introducing technical noise [2, 8]. In
addition, to be compatible with most deep-sequencing technolo-
gies, longer RNAs have to be fragmented into smaller pieces
(200–500 bp) by including a number of steps (RNA fragmentation,
cDNA synthesis, adapter ligation, etc.) that might introduce biases
into the resulting data [5, 8, 9]. RNA-Seq also brings bioinformatic
challenges associated with storage, retrieval, and processing of large
amounts of data. For example, the alignment of long RNA-Seq
reads can be complicated due to nonunique mapping to multiple
locations in the genome. Furthermore, the detection of rare tran-
scripts requires costly deeper sequencing [2].
In addition, high abundance of ribosomal RNA (rRNA) in total
RNA imposes the process of selective sequencing of poly-A-tailed
mRNA transcripts in eukaryotic cells or depletion of rRNA
[8, 10]. Poly(A) selection typically requires a relatively high pro-
portion of mRNA with minimal degradation. However, many
biological samples (such as tissue biopsies) cannot be obtained in
adequate quantity or good mRNA integrity to produce good poly
(A) RNA-seq libraries and therefore require rRNA depletion such
as in the case of salivary rRNA [8, 9].
Although RNA-Seq is still in the early stages of use, it has clear
advantages over previously developed transcriptomic methods such
as microarray profiling [2]. It has a wide variety of applications, but
no single analysis pipeline can be used for all biofluids [8]. Specifi-
cally, RNA-Seq of saliva is challenging, including difficult RNA
isolation step as well as laborious RNA-Seq library construction
stage, inclusion of spike in standards and controls, sequencing of
data, and data storage and analysis [11–13].
The literature about characterization of human salivary extra-
cellular RNA by NGS is scarce [10, 14–16]. Therefore, in our
chapter, we present a protocol for saliva collection, pre- and post-
processing, for RNA-Seq methods.

2 Available Methods for Saliva Collection, Pre- and Post-processing, for RNA-
Sequencing

2.1 Saliva Collection After unstimulated human saliva collection by spitting method, the
samples should be thawed, centrifuged at 2600
g for 15 min at
4  C, stored at 80  C until further analysis, and treated for the
concurrent stabilization of proteins and RNA by the inclusion of a
 RNA Sequencing Analysis of Saliva exRNA 5

protease inhibitor cocktail (aprotinin, PMSF, and sodium orthova-

nadate) and RNase inhibitor (SUPERaselIn; Ambion, Austin, TX)
based on the published saliva standard operating procedure (SOP)
[17, 18].

2.2 RNA Extraction Most of the commercial kits used for RNA isolation provide RNA
Methods yield diluted in big volumes. Thus, if needed small volume for
further analysis, RNA of low concentration prevents adequate
library construction and receiving RNA-Seq reads of good quality.
Therefore, it is essential that all the preparation stages before
sequencing will be performed at the highest level. Specifically, saliva
requires special attention since RNA load in saliva is much lower
than in blood.
Currently, a wide variety of commercial kits are available for
RNA isolation for NGS. They are based on organic extraction,
silica-membrane-based spin column technology, or paramagnetic
particle technology [15]. However, each of these methods has
some advantages as well as disadvantages. For example: the
phenol-Guanidine Isothiocyanate (GITC)-based organic extrac-
tion is often much more contaminated with proteins and other
cellular materials, organic solvents, as well as with DNA, compared
to silica column and paramagnetic particle-based RNA isolation
systems [16]. However, the latter ones can also cause DNA
contamination [15].
In the recent publication, Li et al. [19] compared several com-
mercially available kits for total RNA isolation from cell-free saliva
(CFS) including phenol-based [miRNeasy micro Kit (Qiagen),
TRIzol® Plus RNA Purification Kit (Invitrogen), and mirVana
miRNA Isolation Kit (Ambion)] as well as non-phenol-based kits
[Quick-RNA™ MicroPrep kit (Zymo Research), QIAamp Viral
RNA Mini Kit (Qiagen), and NucleoSpin miRNA (Macherey-
Nagel)]. TRIzol® Plus RNA Purification Kit (Invitrogen) utilizes
organic extraction, whereas QIAamp Viral RNA Mini Kit (Qiagen)
and NucleoSpin miRNA (Macherey-Nagel) are based on silica-
membrane spin column technology. Specifically, the TRIzol® Plus
RNA Purification Kit (Invitrogen) uses Trizol, a GITC-containing
chaotropic lysis-buffer premixed with phenol for precipitation of
nucleic acids from the sample, and uses RNase-free DNase for the
subsequent RNA purification. The miRNeasy micro Kit (Qiagen)
utilizes phenol/guanidine-based lysis of samples and silica-
membrane-based purification of total RNA. In turn, the Quick-
RNA™ MicroPrep kit (Zymo Research) combines a unique buffer
system with Zymo-Spin column, while mirVana miRNA Isolation
Kit (Ambion) employs organic extraction and spin column technol-
ogy (glass fiber filter) [20]. It appeared that the use of the miR-
Neasy Micro Kit (Qiagen) (total RNA yield ¼ 31 ng) and the
NucleoSpin miRNA kit (Macherey-Nagel) (total RNA
yield ¼ 32 ng) seemed to be the best methods for human CFS
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