Methods in Molecular Biology 1537 Gregory J Seymour Mary P Cullinan Nicholas C.K Heng Editors Oral Biology Molecular Techniques and Applications Second Edition Methods in Molecular Biology Series Editor John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Oral Biology Molecular Techniques and Applications Second Edition Edited by Gregory J Seymour Faculty of Dentistry, University of Otago, Dunedin, New Zealand Mary P Cullinan Department of Oral Sciences, Faculty of Dentistry, University of Otago, Dunedin, New Zealand Nicholas C.K Heng Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand Editors Gregory J Seymour Faculty of Dentistry University of Otago Dunedin, New Zealand Mary P Cullinan Department of Oral Sciences, Faculty of Dentistry University of Otago Dunedin, New Zealand Nicholas C.K Heng Faculty of Dentistry, Sir John Walsh Research Institute University of Otago Dunedin, New Zealand ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6683-7 ISBN 978-1-4939-6685-1 (eBook) DOI 10.1007/978-1-4939-6685-1 Library of Congress Control Number: 9781493967384 © Springer Science+Business Media LLC 2017 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made Cover illustration: Example of a bead experiment combined with in situ hybridization (ISH) analysis to study gene expression in embryonic tissue explants The image shows the effects of BMP2 beads on ld1 gene expression in explants of calvarial mesenchyme Photograph provided by D Rice and K Närhi The bead and ISH experiments are described in Chapter 20 Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A Preface It is widely accepted that “evidence-based dentistry” is fundamental to clinical practice and that well-controlled randomized clinical trials followed by systematic reviews and meta- analyses provide much of this evidence base However, it is still the basic biological and physical sciences that underpin advances in dentistry and form the basis for subsequent clinical trials It is equally true that the treatment of any disease should be based on an understanding of the etiology and pathogenesis of that disease, and in this context, the future of dentistry lies very much in continued research in the basic biological sciences This second edition of Oral Biology: Molecular Techniques and Applications continues the approach taken in the first edition and has not attempted to cover all aspects of oral biology, but rather to present a selection of cellular and molecular techniques that can be adapted to cover a range of applications and diseases The first part on saliva, for example, has been updated and expanded to include proteomic analyses by mass spectrometry and NMR-based metabolomics that can be used not only in the study of saliva but also in assessing other oral fluids such as gingival fluid Clearly, saliva is unique to the oral cavity but so too is gingival fluid which, in essence, is the fluid medium of the gingiva and gingival sulcus, and thus is the fluid environment where interactions between the plaque biofilm and the host take place Hence, techniques for its collection and analysis have now been included Although it is years since publication of the first edition of this book, many of the techniques described are still in widespread use and so have been retained, albeit updated, in this second edition In the part on molecular biosciences, for example, chapters on profiling of oral microbial communities, quantitative real-time PCR, and adhesion of yeast and bacteria to oral surfaces have all been retained but substantially updated Epigenetics is now a major theme in biology and is providing great insight into how we interact with our environment As DNA methylation features heavily in epigenetic studies, new chapters on tools and strategies that facilitate the analysis of genome-wide or gene- specific DNA methylation patterns have been included As in the first edition, the last part of this second edition deals with a range of approaches that enable the behavior of cells and tissues in both health and disease to be analyzed at the molecular level The future of dentistry and of the profession lies in research, and it is anticipated that this second edition of Oral Biology: Molecular Techniques and Applications will continue to be a useful resource for oral biologists at all levels, be they students, early career or experienced veterans, and that it provides a ready reference enabling new techniques and approaches to be used in answering a range of specific scientific questions that will underpin a deeper understanding and treatment of oral diseases Dunedin, New Zealand Dunedin, New Zealand Dunedin, New Zealand Gregory J. Seymour Mary P. Cullinan Nicholas C.K. Heng v Contents Preface v Contributors xi Part I Saliva and Other Oral Fluids Salivary Diagnostics Using Purified Nucleic Acids Paul D Slowey RNA Sequencing Analysis of Salivary Extracellular RNA Blanca Majem, Feng Li, Jie Sun, and David T.W Wong Qualitative and Quantitative Proteome Analysis of Oral Fluids in Health and Periodontal Disease by Mass Spectrometry Erdjan Salih Antioxidant Micronutrients and Oxidative Stress Biomarkers Iain L.C Chapple, Helen R Griffiths, Mike R Milward, Martin R Ling, and Melissa M Grant NMR-Based Metabolomics of Oral Biofluids Horst Joachim Schirra and Pauline J Ford Gene Therapy of Salivary Diseases Bruce J Baum, Sandra Afione, John A Chiorini, Ana P Cotrim, Corinne M Goldsmith, and Changyu Zheng 17 37 61 79 107 Part II Molecular Biosciences The Oral Microbiota in Health and Disease: An Overview of Molecular Findings José F Siqueira Jr and Isabela N Rôças Microbial Community Profiling Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE) José F Siqueira Jr., Mitsuo Sakamoto, and Alexandre S Rosado Analysis of 16S rRNA Gene Amplicon Sequences Using the QIIME Software Package Blair Lawley and Gerald W Tannock 10 Adhesion of Yeast and Bacteria to Oral Surfaces Richard D Cannon, Karl M Lyons, Kenneth Chong, Kathryn Newsham-West, Kyoko Niimi, and Ann R Holmes 11 Quantitative Analysis of Periodontal Pathogens Using Real-Time Polymerase Chain Reaction (PCR) Mª José Marin, Elena Figuero, David Herrera, and Mariano Sanz vii 127 139 153 165 191 viii Contents 12 Methods to Study Antagonistic Activities Among Oral Bacteria Fengxia Qi and Jens Kreth 13 Natural Transformation of Oral Streptococci by Use of Synthetic Pheromones Gabriela Salvadori, Roger Junges, Rabia Khan, Heidi A Åmdal, Donald A Morrison, and Fernanda C Petersen 14 Markerless Genome Editing in Competent Streptococci Roger Junges, Rabia Khan, Yanina Tovpeko, Heidi A Åmdal, Fernanda C Petersen, and Donald A Morrison 15 Tools and Strategies for Analysis of Genome-Wide and Gene-Specific DNA Methylation Patterns Aniruddha Chatterjee, Euan J Rodger, Ian M Morison, Michael R Eccles, and Peter A Stockwell 16 Generating Multiple Base-Resolution DNA Methylomes Using Reduced Representation Bisulfite Sequencing Aniruddha Chatterjee, Euan J Rodger, Peter A Stockwell, Gwenn Le Mée, and Ian M Morison 17 A Protocol for the Determination of the Methylation Status of Gingival Tissue DNA at Specific CpG Islands Trudy J Milne 18 Genome-Wide Analysis of Periodontal and Peri-Implant Cells and Tissues Moritz Kebschull, Claudia Hülsmann, Per Hoffmann, and Panos N Papapanou 19 Differential Expression and Functional Analysis of High-Throughput -Omics Data Using Open Source Tools Moritz Kebschull, Melanie Julia Fittler, Ryan T Demmer, and Panos N Papapanou 20 Exploring Genome-Wide Expression Profiles Using Machine Learning Techniques Moritz Kebschull and Panos N Papapanou 203 219 233 249 279 299 307 327 347 Part III Cells and Tissues 21 Embryonic Explant Culture: Studying Effects of Regulatory Molecules on Gene Expression in Craniofacial Tissues 367 Katja Närhi 22 Oral Epithelial Cell Culture Model for Studying the Pathogenesis of Chronic Inflammatory Disease 381 Mike R Milward, Martin R Ling, Melissa M Grant, and Iain L.C Chapple 23 Fabrication and Characterization of Decellularized Periodontal Ligament Cell Sheet Constructs 403 Amro Farag, Cédryck Vaquette, Dietmar W Hutmacher, P Mark Bartold, and Saso Ivanovski Contents 24 A Method to Isolate, Purify, and Characterize Human Periodontal Ligament Stem Cells Krzysztof Mrozik, Stan Gronthos, Songtao Shi, and P Mark Bartold 25 Constructing Tissue Microarrays: Protocols and Methods Considering Potential Advantages and Disadvantages for Downstream Use Lynne Bingle, Felipe P Fonseca, and Paula M Farthing 26 Growing Adipose-Derived Stem Cells Under Serum-Free Conditions Diogo Godoy Zanicotti and Dawn E Coates 27 Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts Dawn E Coates, Sobia Zafar, and Trudy J Milne 28 Proteomic Analysis of Dental Tissue Microsamples Jonathan E Mangum, Jew C Kon, and Michael J Hubbard 29 Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps Phillipa C White, Ilaria J Chicca, Martin R Ling, Helen J Wright, Paul R Cooper, Mike R Milward, and Iain L.C Chapple ix 413 429 439 447 461 481 Index 499 Contributors Sandra Afione • Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda, MD, USA Heidi A. Åmdal • Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway P. Mark Bartold • Colgate Australian Clinical Dental Research Centre, Dental School, University of Adelaide, Adelaide, Australia Bruce J. Baum • Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda, MD, USA; Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda, MD, USA Lynne Bingle • Academic Unit of Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, UK Richard D. Cannon • Department of Oral Sciences, University of Otago School of Dentistry, Dunedin, New Zealand; Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago School of Dentistry, Dunedin, New Zealand Iain L.C. Chapple • School of Dentistry, Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Aniruddha Chatterjee • Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand Ilaria J. Chicca • Institute of Clinical Sciences, College of Medical and Dental Sciences, The School of Dentistry, University of Birmingham, Birmingham, UK John A. Chiorini • Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda, MD, USA Kenneth Chong • Department of Oral Sciences, University of Otago School of Dentistry, Dunedin, New Zealand Dawn E. Coates • Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand Paul R. Cooper • Institute of Clinical Sciences, College of Medical and Dental Sciences, The School of Dentistry, University of Birmingham, Birmingham, UK Ana P. Cotrim • Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda, MD, USA Ryan T. Demmer • Department of Epidemiology, Columbia University Mailman School of Public Health, New York, NY, 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 Amro Farag • School of Dentistry and Oral Health, Regenerative Medicine Center, Menzies Health Institute Queensland, Gold Coast, QLD, Australia xi xii Contributors Paula M. Farthing • Academic Unit of Oral and Maxillofacial Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield, UK Elena Figuero • Oral Research Laboratory, Faculty of Odontology, University Complutense, Madrid, Spain; Etiology and Therapy of Periodontal Diseases (ETEP) Research Group, University Complutense, Madrid, Spain; Department of Periodontology, Faculty of Dentistry, University Complutense of Madrid, Madrid, Spain Melanie Julia Fittler • Department of Periodontology, Operative and Preventive Dentistry, University of Bonn, Bonn, Germany Felipe P. Fonseca • Department of Oral Diagnosis, Faculty of Dentistry of Piracicaba, FOP, UNICAMP, Piracicaba, São Paolo, Brazil Pauline J. Ford • School of Dentistry, Oral Health Centre, The University of Queensland, Herston, QLD, Australia Corinne M. Goldsmith • Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health (NIH), Bethesda, MD, USA Melissa M. Grant • School of Dentistry, Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Helen R. Griffiths • School of Dentistry, Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Stan Gronthos • Mesenchymal Stem Cell Group, Adelaide Medical School, Faculty of Health Sciences, University of Adelaide, Adelaide, SA, Australia David Herrera • Etiology and Therapy of Periodontal Diseases (ETEP) Research Group, University Complutense, Madrid, Spain; Department of Periodontology, Faculty of Dentistry, University Complutense of Madrid, Madrid, Spain Per Hoffmann • Department of Genomics, Institute of Human Genetics, University of Bonn, Bonn, Germany; Human Genomics Research Group, Department of Biomedicine, University of Basel, Basel, Switzerland Ann R. Holmes • Department of Oral Sciences, University of Otago School of Dentistry, Dunedin, New Zealand; Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago School of Dentistry, Dunedin, New Zealand Michael J. Hubbard • Department of Pharmacology and Therapeutics, University of Melbourne, Melbourne, VIC, Australia; Department of Pediatrics, Royal Children’s Hospital, University of Melbourne, Melbourne, VIC, Australia Claudia Hülsmann • Department of Periodontology, Operative and Preventive Dentistry, Faculty of Medicine, University of Bonn, Bonn, Germany Dietmar W. Hutmacher • Queensland University of Technology, Brisbane, QLD, Australia Saso Ivanovski • School of Dentistry and Oral Health, Regenerative Medicine Center, Menzies Health Institute Queensland, Gold Coast, QLD, Australia; Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia Roger Junges • Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway Moritz Kebschull • Department of Periodontology, Operative and Preventive Dentistry, Faculty of Medicine, University of Bonn, Bonn, Germany; Division of Periodontics, Section of Oral, Diagnostic and Rehabilitation Sciences, Columbia University College of Dental Medicine, New York, NY, USA Rabia Khan • Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway 488 Phillipa C White et al 3.4 Quantification of NET-Bound Components 3.4.1 Production and Storage of NETs Add mL syringe-filtered % BSA to each well of a clear 24-well plate and store at °C Following neutrophil isolation the next day, remove the % BSA from the plate using an aspirator (see Note 10) Add × 106 neutrophils in 875 μL RPMI-1640 to each well and incubate for a 30 baseline period (37 °C, % CO2) (see Note 11) Stimulate neutrophils in 125 μL aliquots with PBS (negative control), PMA (50 nM), HOCl (0.75 mM), or bacteria (1 × 108, MOI of 1000), cover with foil and incubate for h (37 °C, % CO2) (See Notes 12–14) Post-incubation wash the NETs by gently aspirating the supernatant and adding mL of pre-warmed RPMI-1640 Repeat these steps once more (See Notes 19 and 20) Add 75 μL of MNase at unit/mL to each well and incubate for 15 at room temperature Centrifuge the plate for 10 at 1800 × g (see Note 15) Post-centrifugation, NET bound components can be quantified immediately or at a later date (see Note 21) 3.4.2 Measuring NET-Bound Neutrophil Elastase (NE) Add 100 μL of NET supernatant to each well of a clear 96-microwell plate in duplicate Add 100 μL of 0.5 M N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide to each well, cover the plate with foil, and incubate for h (37 °C, % CO2) Post-incubation, measure the absorbance at 405 nm Generate a standard curve by serially diluting human NE in 100 μL aliquots and running in duplicate on the same plate 3.4.3 Measuring NET-Bound Myeloperoxidase (MPO) Add 50 μL of NET supernatant to each well of a clear 96-well plate in duplicate Add 50 μL of TMB substrate solution to each well, cover the plate with foil, and incubate for 20 at room temperature Post-incubation, add 50 μL of M sodium phosphate to stop the reaction Measure the absorbance at 450 nm Generate a standard curve by serially diluting human MPO in 50 μL aliquots and running in duplicate on the same plate 3.4.4 Measuring NET-bound cathepsin G (CG) Add 50 μL of NET supernatant to each well of a clear 96-well plate in duplicate Add 50 μL of mM N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide in 0.1 M HEPES to each well, cover the plate with foil, and incubate for h (37 °C, % CO2) Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps 489 Post-incubation, measure the absorbance at 405 nm Generate a standard curve by serially diluting human CG in 50 μL aliquots and running in duplicate on the same plate 3.5 Quantification of NET Entrapment of Bacteria Add 200 μL syringe-filtered % BSA to each well of a black 96-well plate and store at °C overnight Following neutrophil isolation the next day, remove the % BSA from the plate using an aspirator (see Note 10) Add × 105 neutrophils in 175 μL RPMI-1640 to each well and incubate for a 30 baseline period (37 °C, % CO2) (see Note 11) Stimulate neutrophils with 25 μL of HOCl (0.75 mM); also include a neutrophil-free well and cells treated with PBS as negative controls Cover with foil and incubate for h (37 °C, % CO2) (see Note 14) Following the culture of planktonic bacteria (see Table 1), measure the turbidity by spectroscopy and dilute to × 107 per 50 μL (MOI of 100) with PBS in mL aliquots Fluorescently stain bacteria by incubating bacteria with 0.3 mg/mL FITC for 30 on ice with continuous agitation Next, centrifuge the bacteria (10 at 5000 rpm) to precipitate FITC-stained bacteria, and resuspend in mL PBS Prior to adding FITC-stained bacteria to the well, add unit/ mL MNase to selected wells containing NETs and incubate for 15 at room temperature (see Note 22) Add the live FITC-stained bacteria to the NETs plate in 50 μL aliquots (bacteria added to neutrophil-free wells, PBS-treated cells, HOCl-treated [NETs] cells, and degraded HOCl-NETs) and incubate for h (37 °C, % CO2) Post-incubation, carefully remove the supernatant and replace with 200 μL fresh, pre-warmed RPMI-1640 Repeat wash step (see Note 23) Quantify entrapped bacteria by measuring the fluorescence using a fluorometer (excitation: 485 nm, emission: 535 nm) 3.6 Quantification of NET Killing of Bacteria Add 500 μL syringe-filtered % BSA to each well of a clear 24-well plate and store at °C overnight Following neutrophil isolation the next day, remove the % BSA from the plate using an aspirator (see Note 10) Add × 105 neutrophils in 440 μL RPMI-1640 to each well and incubate for a 30 baseline period (37 °C, % CO2) (see Note 11) Stimulate neutrophils with 60 μL of HOCl (0.75 mM), cover with foil, and incubate for h (37 °C, % CO2) (see Note 14) 11827 43541 10790 10556 35037 27335 33397 10558 33624 (27) Propionybacterium acnes Selenomonas noxia Veillonella parvula Streptococcus sanguinis Streptococcus oralis Streptococcus intermedius Streptococcus anginosus Streptococcus gordonii Capnocytophagia gingivalis Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar % CO2 Liquid media: brain heart infusion Solid media: horse blood agar % CO2 Liquid media: brain heart infusion Solid media: horse blood agar % CO2 Liquid media: brain heart infusion Solid media: horse blood agar % CO2 Liquid media: brain heart infusion Solid media: horse blood agar % CO2 Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion 43718 1.62 × 109 1.69 × 109 1.69 × 109 1.69 × 109 1.69 × 109 1.69 × 109 6.8 × 109 1.69 × 109 1.69 × 109 6.8 × 109 8.3 × 108 Bacteria per mL Growing conditions (37 °C) if OD600nm = Aggregatibacter actinomycetemcomitans serotype b Culture medium Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion ATCC number Actinomyces viscosus (naeslundii genospecies 2) 43146 Bacteria strain Table Culture conditions for periodontal bacterial species 490 Phillipa C White et al 29523 33612(4) 27823 (M32b) 33238 (371) 51146 25586 10953 W83 Aggregatibacter actinomycetemcomitans serotype a Capnocytophaga sputigena Streptococcus constellatus Camylobacter rectus Campylobacter showae Fusobacterium nucleatum sp Nucleatum Fusobacterium nucleatum sp Polymorphum Porphyromonas gingivalis Solid media: fastidious blood with Anaerobic neomycin Liquid media: fastidious Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar % CO2 Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion Solid media: horse blood agar Anaerobic Liquid media: brain heart infusion 1.69 × 109 1.62 × 109 1.62 × 109 6.8 × 109 6.8 × 109 1.69 × 109 1.62 × 109 6.8 × 109 6.8 × 109 Bacteria we have previously used in the above assays are listed alongside their respective ATCC number The agar media, broth, and incubation culturing conditions are outlined below Following planktonic growth, bacterial concentrations were determined by spectrophotometry (OD600nm) and our own experiments determined the number of bacteria per mL of broth if the OD is 1.0 ATCC American Type Culture Collection 23834 Eikenella corrodens Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps 491 492 Phillipa C White et al Next, carefully remove the supernatant from each well and replace with 500 μL pre-warmed RPMI-1640, with or without 10 μg/mL cytochalasin B or unit/mL MNase, incubate for 15 at room temperature (see Note 24) Following the culture of planktonic bacteria (see Table 1), measure the turbidity by spectroscopy and dilute to × 107 per 50 μL (MOI of 100) with PBS in mL aliquots Add the live bacteria in 50-μL aliquots to the selected wells and centrifuge the plate for 10 at 700 × g, then incubate the plate for h (37 °C, % CO2) (see Note 15) Post-incubation, add MNase in 25 μL aliquots (100 unit/mL) and incubate for 15 at room temperature (see Note 25) Transfer the well contents to 1.5-mL centrifuge tubes and dilute with broth To determine bacteria viability, add 50 μL of this suspension and inoculate an agar plate to enumerate bacterial colonies in 24 h (see Note 26) 3.7 Quantification of NET Degradation by Human Plasma Stimulate for NETs with 0.75 mM HOCl in a 96-well plate, as previously described Defrost previously isolated plasma samples at room temperature and dilute to 10 % in PBS Add 10 % plasma to the wells in 50 μL aliquots and incubate for h (37 °C, % CO2) (see Note 27) Treat selected wells with unit/mL MNase for 15 at room temperature, instead of plasma (see Note 28) Following incubation with plasma, centrifuge the plate (10 at 1800 × g), and then transfer 150 μL of the supernatant to a black 96-well plate (see Notes 15 and 16) Add 15 μL of 10 μM Sytox green to quantify any DNA within the supernatant on a fluorometer (excitation: 485 nm, emission: 535 nm) 3.8 Fluorescence Visualization of NETs Add 300 μL of syringe-filtered % BSA to each well of a clear 24-well plate and store at °C overnight Following neutrophil isolation the next day, remove the % BSA from the plate using an aspirator (see Note 10) Add × 105 neutrophils in 260 μL RPMI-1640 to each well and incubate for a 30 baseline period (37 °C, % CO2) (see Note 11) Stimulate neutrophils in 40-μL aliquots with PBS (negative control), PMA (50 nM) or HOCl (0.75 mM), cover with foil, and incubate for h (37 °C, % CO2) (see Notes 14 and 17) Add 30 μL of 10 μM Sytox green and visualize immediately under a fluorescence microscope (excitation: 485 nm, emission: 535 nm) Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps 3.9 Scanning Electron Microscopy (SEM) of NETs 493 Sterilize round 11-mm glass coverslips (see Note 29) in 0.2 M HCl, followed by two wash steps in dH2O Once dry, add 100 μL of syringe-filtered % BSA and keep at room temperature for h prior to use (see Note 10) Add × 105 neutrophils in 100 μL RPMI-1640 to each coverslip and after a 30-min baseline incubation period (37 °C, % CO2) (see Note 11), stimulate cells with PBS (negative control), PMA (50 nM), or live bacteria (1 × 107 MOI of 100) (see Note 14) Following a 4-h incubation (37 °C, % CO2), fix samples with 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 30 at room temperature Dehydrate samples by immersing in a graded ethanol series (20, 30, 40, 50, 60, 70, 90, 100, and 100 % for 10 each) Dry samples by adding 100 μL of HMDS, and leave to evaporate in a fume hood overnight Mount samples onto 25-mm aluminum stubs with carbon conductive tabs and coat in gold for 90 s, prior to analysis by SEM 3.10 Immunos taining of NETs Following the production of NETs, centrifuge the plate (10 at 1800 × g) Postcentrifugation, add 50 μL 20 % PFA to each well and incubate for 10 at room temperature Next, gently remove the media and wash twice with PBS Remove PBS and add 50 μL of 0.1 % PBS-BSA to each well Incubate for 30 at room temperature and wash with PBS Dilute mouse antihuman MPO and rabbit antihuman NE primary antibodies 1/150 in 0.05 % Digitonin PBS Add 50 μL 0.05 % Digitonin PBS plus antibodies to each well and incubate for h at room temperature, then wash with PBS Dilute secondary antibodies goat anti-rabbit IgG Alexa Fluor 594 (Excitation: 590 nm, Emission: 617 nm) and goat antimouse IgG Alexa Fluor 488 (Excitation: 495 nm, Emission: 519 nm) 1/500 in PBS/Hoechst Add 50 μL secondary antibodies in PBS/Hoechst to each well and incubate for 30 at room temperature and then wash with PBS Visualize NETs using a fluorescence microscope 3.11 Bacterial Culture Store bacterial suspensions at −80 °C in cryotubes containing TSB and 10 % DMSO When required, defrost at room temperature and inoculate agar plates by adding 100 μL of bacterial suspension and spreading with a disposable presterilized loop (see Table 1) 494 Phillipa C White et al Loosely wrap plates in cling film, invert and incubate in the appropriate conditions for at least days (see Note 30) Grow cultures planktonically by inoculating a single colony into broth, and incubate for at least days on a shaker (see Notes 31 and 32) Confirm planktonic growth by measuring the optical density in a cuvette (600 nm) using noninoculated media to calibrate the spectrophotometer Centrifuge the bacterial suspension in broth (15 min, 1800 × g, °C) Discard the supernatant and resuspend the bacterial pellet in PBS Repeat centrifugation and PBS wash steps twice more (see Note 33) To heat-kill bacteria, incubate the bacterial suspension in PBS at 80 °C in a microbiology oven for 30 (see Note 34) Notes Collect blood in lithium-heparin anti-coagulated tubes Experiments from our own laboratory reveal greater cell retrieval and less inadvertent cell activation compared with other anticoagulants Isolate cells as soon as possible We typically isolate approximately × 106 cells per mL of blood If possible, prepare gradients immediately prior to neutrophil isolation to minimize chance of gradient disruption; however, gradients may be carefully stored at °C for a day if needed It is more reliable to layer the 1.098 g/mL Percoll underneath the 1.079 g/mL Percoll solution Collect as few erythrocytes as possible to minimize the risk of erythrocyte contamination Erythrocyte lysis is indicated by the increased transparency of the blood/lysis buffer solution Do not incubate neutrophils in erythrocyte lysis buffer for longer than recommended due to the potential for cell activation If erythrocytes are taking time to lyse (solution to go clear) this could be due to the age of the lysis buffer, which should be stored for a maximum of weeks at °C During the lysis buffer/PBS washing stages and removal of supernatant, the operator must take care not to disturb the cell pellet to maximize cell retrieval We recommend duplicate cell counting using a haemocytometer and calculation of the mean We routinely ensure neutrophil viability by trypan blue exclusion and CellTiter-Glo® assays, as well as neutrophil purity by flow cytometry [23] Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps 495 10 Adding % BSA to plasticware prior to the addition of neutrophils coats the surface, which has been found to reduce inadvertent neutrophil activation 11 A 30-min baseline period prior to neutrophil activation allows the cells to settle in the plastic-ware and neutrophils are more responsive to exogenous stimulation 12 HOCl and PMA stimuli are light-sensitive Make up/defrost at room temperature immediately prior to use and protect from light by foil wrapping 13 Following their release, NETs are attached to the neutrophil they are derived from The addition of MNase degrades the NET structures, which following centrifugation and the sedimentation of neutrophils, allows for the fluorometric quantification of NET-DNA in the supernatants 14 NETs are produced in response to HOCl in as little as 30 [9], PMA and bacteria typically induce NET production in and h, respectively 15 Following MNase digestion, neutrophils are centrifuged in a plate spinner If you not have a plate spinner then the well contents can be transferred to 1.5-mL centrifuge tubes for centrifugation 16 Care needs to be taken when transferring NET supernatants to a new plate to ensure the operator is not disturbing the neutrophils at the bottom of the well; tipping the plate toward you helps 17 Bacteria produce DNA [24] that can interfere with NET-DNA assays If quantifying NET production in response to bacteria, control wells containing bacteria in PBS (and no neutrophils) need to be measured and subtracted from the final NET production values We not employ a bacterial NET stimulus during Sytox green fluorescence microscopy as it is very difficult to distinguish between NET-DNA and bacterial DNA; therefore, use PMA or HOCl for NET fluorescence visualization 18 Sytox green does not permeate viable cells; therefore, very high readings (DNA) in control wells are indicative of high numbers of nonviable neutrophils 19 Neutrophils and the attached NET structures settle to the bottom of plastic-ware; however, they are not adherent cells and washing needs to be careful to minimize the disruption of NET structures 20 Washing removes components that are not NET-bound but are concurrently released during neutrophil activation 21 NET-bound components can be quantified immediately, or transferred to cryotubes and stored at °C (for up to week) or at −20 °C (for up to months) for quantification at a later date 496 Phillipa C White et al 22 The addition of DNase to NETs in selected wells produces degraded NET structures to serve as an additional control 23 The duplicate wash steps ensure bacteria not entrapped within NET structures are removed and therefore only the fluorescence of entrapped bacteria is measured 24 The addition of cytochalasin B to selected wells following NET release inhibits neutrophil phagocytosis and the addition of MNase degrades NETs 25 The addition of MNase postincubation disassembles the NETs and frees the bacteria prior to inoculation on agar plates 26 The dilution factor required prior to inoculating agar plates with bacteria needs to be determined in preliminary experiments for each bacterial species 27 Plasma is isolated from whole blood in mL lithium heparin anti-coagulant tubes by centrifuging for 30 at 1000 × g (4 °C) Following centrifugation, plasma is transferred to cryotubes in 500-μL aliquots and stored at −80 °C 28 The treatment of some wells with MNase will serve as a positive control and the MNase treatment is considered 100 % NET degradation 29 It is easier to handle coverslips for SEM use in 6-well plates 30 Wrapping agar plates in clingfilm helps to prevent the desiccation of agar following prolonged incubation 31 Periodontal bacteria typically take at least days to grow planktonically However, growth can be determined at any time point following inoculation of broth by assessing the turbidity, as determined by measuring the optical density at 600 nm 32 Confirm the bacterial species by gram staining and/or PCR 33 Planktonic bacteria are washed in PBS multiple times to remove broth prior to neutrophil assays 34 Killing of bacteria can be confirmed by inoculating a fresh agar plate with the heated bacteria suspension References Borregaard N, Cowland JB (1997) Granules of the human neutrophilic polymorphonuclear leukocyte Blood 89:3503–3521 Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A (2004) Neutrophil extracellular traps kill bacteria Science 303: 1532–1535 Bianchi M, Niemiec MJ, Siler U, Urban CF, Reichenbach J (2011) Restoration of antiAspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent J Allergy Clin Immunol 127:1243–1252 Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, Hayama R, Leonelli L, Han H, Grigoryev SA, Allis CD, Coonrod SA (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation J Cell Biol 184:205–213 Yousefi S, Mihalache C, Kozlowski E, Schmid I, Simon HU (2009) Viable neutrophils release Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps 10 11 12 13 14 mitochondrial DNA to form neutrophil extracellular traps Cell Death Differ 16:1438–1444 Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, Sibley CD, Robbins SM, Green FH, Surette MG, Sugai M, Bowden MG, Hussain M, Zhang K, Kubes P (2010) A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus J Immunol 185:7413–7425 Harris PC (2012) Effect of density gradient material upon ex-vivo neutrophil behaviour, and effect of neutrophil extracellular traps upon the growth and survival of periodontopathogenic bacteria MRes thesis, University of Birmingham Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A (2007) Novel cell death program leads to neutrophil extracellular traps J Cell Biol 176:231–241 Palmer L, Cooper PR, Ling MR, Wright HJ, Huissoon A, Chapple IL (2012) Hypochlorous acid regulates neutrophil extracellular trap release in humans Clin Exp Immunol 167:261–268 Beiter K, Wartha F, Albiger B, Normark S, Zychlinsky A, Henriques-Normark B (2006) An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps Curr Biol 16:401–407 Delbosc S, Alsac JM, Journe C, Louedec L, Castier Y, Bonnaure-Mallet M, Ruimy R, Rossignol P, Bouchard P, Michel JB, Meilhac O (2011) Porphyromonas gingivalis participates in pathogenesis of human abdominal aortic aneurysm by neutrophil activation Proof of concept in rats PLoS One 6, e18679 Behrendt JH, Ruiz A, Zahner H, Taubert A, Hermosilla C (2010) Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis Vet Immunol Immunopathol 133:1–8 Byrd AS, O’Brien XM, Johnson CM, Lavigne LM, Reichner JS (2013) An extracellular matrix–based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans J Immunol 190:4136–4148 Saitoh T, Komano J, Saitoh Y, Misawa T, Takahama M, Kozaki T, Uehata T, Iwasaki H, Omori H, Yamaoka S, Yamamoto N, Akira S (2012) Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1 Cell Host Microbe 12:109–116 497 15 Keshari RS, Jyoti A, Dubey M, Kothari N, Kohli M, Bogra J, Barthwal MK, Dikshit M (2012) Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition PLoS One 7, e48111 16 Palmer LJ (2010) Neutrophil extracellular traps in periodontitis Ph.D thesis, University of Birmingham 17 Berends ET, Horswill AR, Haste NM, Monestier M, Nizet V, von Köckritz-Blickwede M (2010) Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps J Innate Immun 2:576–586 18 Wartha F, Beiter K, Albiger B, Fernebro J, Zychlinsky A, Normark S, Henriques-Normark B (2007) Capsule and D‐alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps Cell Microbiol 9:1162–1171 19 Lauth X, von Köckritz-Blickwede M, McNamara CW, Myskowski S, Zinkernagel AS, Beall B, Ghosh P, Gallo RL, Nizet V (2009) M1 protein allows Group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition J Innate Immun 1:202–214 20 Urban CF, Reichard U, Brinkmann V, Zychlinsky A (2006) Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms Cell Microbiol 8:668–676 21 Hakkim A, Fürnrohr BG, Amann K, Laube B, Abed UA, Brinkmann V, Herrmann M, Voll RE, Zychlinsky A (2010) Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis Proc Natl Acad Sci U S A 107:9813–9818 22 Leffler J, Martin M, Gullstrand B, Tydén H, Lood C, Truedsson L, Bengtsson AA, Blom AM (2012) Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease J Immunol 188:3522–3531 23 Zhou L, Somasundaram R, Nederhof RF, Dijkstra G, Faber KN, Peppelenbosch MP, Fuhler GM (2012) Impact of human granulocyte and monocyte isolation procedures on functional studies Clin Vaccine Immunol 19:1065–1074 24 Das T, Sehar S, Manefield M (2013) The roles of extracellular DNA in the structural integrity of extracellular polymeric substance and bacterial biofilm development Environ Microbiol Rep 5:778–786 Index A Adeno-associated virus�����������������������������������������������������108 Adenovirus��������������������������������������������������������������� 109, 110 Adherence isolation��������������������������������������������������419–420 Adhesion of bacteria to saliva-coated dentures���������������������������167 of bacteria to saliva-coated hydroxyapatite������������������������� 167, 169–170, 177 of bacteria to saliva-coated medical-grade silicone������������������������������������ 167, 171, 179–180 of saliva-coated yeast to epithelial cells�����������������������167 of yeast to immobilized proteins��������� 168–169, 174–177 of yeast to saliva-coated denture prostheses����������������167 of yeast to saliva-coated hydroxyapatite������������������������� 167, 169–170, 177 of yeast to saliva-coated medical-grade silicone������������������������������������ 167, 171, 179–180 Adipose-derived stem cells (ADSC)������������������������439–444 Aggregatibacter actinomycetemcomitans���������������������� 133, 191, 198, 490, 491 AllPrep® DNA/RNA/Protein kit�����������������������������300–302 Allprotect™ tissue reagent������������������������������� 300, 301, 305 Ameloblastin����������������������������������������������������������������������375 Animal models mouse�������������������������������������������������������������������������115 rat�������������������������������������������������������������������������������113 Antibody��������������������������������������������������6, 38, 69, 251, 254, 383, 384, 388, 392, 394, 406, 410, 415–422, 424, 425, 437, 471, 493 Antioxidant������������������������������������������������������������������61–76 Ascorbic acid��������������������������������������������62, 64, 67, 74, 370, 377, 404, 407, 448, 451 B Bacterial DNA cloning�������������������������������������������������������� 128, 206, 212 expression vector���������������������������������������������������������215 preparation���������������������������������������������������������� 197, 495 Bacteriocin activity assay���������������������������������������������������������������209 competition assay (agar plate)�������������������������������������207 competition assay (biofilm)���������������������������������207–208 derivatization������������������������������������������������������ 206, 211 isolation of structural genes��������������������������������211–213 N-terminal sequencing������������������������� 53, 210, 211, 216 purification��������������������������������������������������������� 206, 210 Biofilm bacteriocin competition assay�����������������������������207–208 confocal laser scanning microscopy����������������������������205 Bioinformatics�����������������������������������154, 160, 266, 270, 340 Biomarkers�������������4, 17, 18, 37, 38, 61–76, 80, 98, 100, 429 Bisphosphonate-related osteonecrosis of the jaw (BRONJ)��������������������������������������������������������448 Bisulfite sequencing����������������������������������������� 250, 252, 254, 260–262, 266, 267, 270, 279–294 C Calvarial bone������������������������������������������� 368, 369, 373, 377 Campylobacter rectus�������������������������������������������������� 193, 198 Candida albicans adhesion to epithelial cells������������������������������������������381 adhesion to saliva-coated dentures������������������������������167 adhesion to saliva-coated hydroxyapatite������������������������� 167, 169–170, 177 adhesion to saliva-coated medical-grade silicone����������������������������������������������������179–180 genomic dna purification��������������������������������������������154 Cathepsin G����������������������������������������������������������� 485, 488–489 Cell sheet������������������������������������������������������������������403–411 Chemiluminescence���������������������������������������������������������487 Cloning in Escherichia coli����������������������������������������� 188, 268 Colonization������������������������������ 127, 133, 165, 166, 192, 204 Comet assay������������������������������������������������ 62, 66, 69–71, 75 Competence (for genetic transformation)������������������������233 Competence-stimulating peptide (CSP) of anginosus group streptococci������������������������� 219–221, 227, 228, 490 of mitis group streptococci������������������������� 130, 219–221, 223, 225–228 of mutans group streptococci����������������������������� 221, 223, 224, 230, 234 Complementary DNA (cDNA) synthesis�������� 369, 372, 376 Complementary RNA (cRNA)������������������������������������������17 Computational genomics������������������������������������������ 253, 348 Confocal laser scanning microscopy (CLSM)��������������������205 CpG islands��������������� 252, 279, 280, 291, 299–305, 333, 350 CSP See Competence-stimulating peptide (CSP) Gregory J Seymour et al (eds.), Oral Biology: Molecular Techniques and Applications, Methods in Molecular Biology, vol 1537, DOI 10.1007/978-1-4939-6685-1, © Springer Science+Business Media LLC 2017 499 Oral Biology: Molecular Techniques and Applications 500 Index D Decellularization�������������������������������������� 404, 405, 409–411 Delivery devices See Viral vectors Denaturing gradient gel electrophoresis DNA extraction������������������������������������������� 62, 140–143, 194, 196–197, 200, 282 GC clamp����������������������������������������������������������� 140, 141 Dental development���������������������������������������������������������461 DGGE See Denaturing gradient gel electrophoresis Differential expression analysis�������������������������������� 327–328, 337–339, 343, 348, 357 Differential methylation������������������� 257, 260–263, 291–292 Differential methylation analysis package (DMAP)������������������ 255, 257, 261, 262, 291, 292 DNA purification from Candida albicans (and related species)���������������229, 282, 315 from Porphyromonas gingivalis��������������������������� 191, 193, 197, 198, 381 from Streptococcus spp.������������������132, 133, 135, 225, 227 E Elastase��������������������������������������������������������������������� 485, 488 Embryonic tissue explants culture�����������������������������������������������������������������367–379 dissection���������������������������������������������������� 370–371, 373 fixation������������������������������������������������ 166, 371, 373–375 In situ hybridization (ISH)����������������� 369, 371–372, 375 mouse������������������������������������������������� 368, 371, 373, 378 Enamel epithelium������������������������������������462, 465, 467, 474, 476 matrix����������������������������������������������������������������� 462, 467 microdissection��������������������������������������������������� 462, 467 protein extraction�������������������������������� 462, 465, 467–468 Enamel defects��������������������������������������������������������� 462, 473 Epidemiology�����������������������������������������������������������������������4 Epigenetics������������������������ 249, 260, 263, 265, 270, 279, 299 Epithelial cells��������������������������������������5, 107, 108, 167, 170, 177–179, 381–383, 385–392, 394–397, 400, 401 Epithelium and mesenchyme interactions���������������� 367, 378 Exosomes���������������������������������������������������������� 5–6, 9–11, 13 Extracellular RNAs (exRNAs)������������������������� 17, 18, 20–33 F FACS sorting����������������������������������������������������������������� 416, 422 Fetal bovine serum (FBS)�������������������������������� 114, 310, 321, 369, 415, 439, 448 Flow cytometry��������������������������������������������������������421–422 Fluorescence����������������������������������� 34, 64, 67, 128, 144, 179, 192, 193, 200, 208, 269, 325, 384, 395, 415–416, 454, 487, 489 Fluorescence microscopy�������������������482, 485, 492, 493, 495 Functional proteomics������������������������������������������������������414 Fusobacterium spp.����������������������131–134, 145, 193–195, 198 G Gel electrophoresis�����������������������������������������46, 49, 50, 122, 235, 238, 239, 292, 304, 385, 389, 397, 399, 465, 470–471 Gel preparation (protein)�������������������������� 187, 463, 468–470 Gene expression analysis�������������������215, 269, 369, 375, 377 Luciferase assay����������������������������������������������������������215 Microarrays��������������������������������������������������������� 308, 309 qPCR��������������������������������������������������269, 324, 369, 372, 376–377, 450, 454 Reporter gene����������������������������������������������������� 215, 378 Gene therapy������������������������������������������������������������107–122 Genomic DNA�����������������������������������������5, 6, 154, 226, 229, 251, 252, 268, 280, 282, 294, 301–304, 306, 318, 320, 396, 449, 454 Geranylgeraniol����������������������������������������������������������������449 Gingiva cells�������������������������������������������������������������������������64, 72 tissue���������������������������������������������������� 82, 299–325, 330, 339, 348, 349, 359 Gingival crevicular fluid (GCF)���������������������������� 38, 40, 42, 43, 50–55, 64, 67, 72–73, 80, 83, 84, 191, 194 Gingival tissue������������������������������������������� 82, 299–305, 308, 309, 317, 324, 330, 339, 348, 349, 359 H High-performance liquid chromatography (HPLC)������������������������������������������������������64, 67 High-throughput DNA sequencing (HTS)���������������� 4, 135, 153, 268–269, 292, 311–312, 318–320, 327–343, 384, 385, 391, 393, 394, 429 Histology������������������������������������������������������������������ 371, 375 Homologous recombination����������������������������� 213, 215, 233 H400 oral epithelial cells��������������������������������������������������386 HPLC See High-performance liquid chromatography (HPLC) HumanMethylation450 BeadChip�������������������������� 252, 258 Hydrogen peroxide (H2O2) assay������������������ 65, 69, 204, 424 Hydroxyapatite��������������������������167, 169–170, 177, 417, 423 I Immunocytochemistry���������������������������������������������� 383, 388 Immunohistochemistry���������������������417–418, 424, 430, 437 Immunohistology Immunomagnetic bead separation����������������������������415–416 Immunostaining������������������������405–406, 409–410, 483, 493 In situ hybridization���������������������������������� 369, 371–372, 375 Interspecies competition����������������������������������� 207, 208, 213 Oral Biology: Molecular Techniques and Applications 501 Index K 450K See HumanMethylation450 BeadChip L Luciferase reporter������������������������������������������������������������215 M Markerless mutagenesis����������������������������������� 223, 229, 230, 238–239 Mass spectrometry (MS)����������������������������� 4, 37–46, 48–58, 67, 79, 100, 102, 210, 471, 472, 475 Mesenchymal stem cells (MSC)������������������������������ 414, 418, 419, 422, 439, 441 Metabolomics���������������������������������������������������������������������83 Microarrays Affymetrix���������������������������������������������������������� 340, 353 data analysis���������������������������� 18, 97, 102, 160, 198, 250, 256, 259, 260, 266, 267, 270, 296, 305, 450, 454 real-time PCR, 128, 129, 300 Microbial community profiling��������������������������������139–151 Microbial ecology�������������������������������������������������������������153 Microdissection����������������������������������������������������������������252 enamel epithelium���������������������������������������������� 462, 467 enamel matrix����������������������������������������������������� 462, 467 Micronutrients��������������������������������������������������������������61–76 MicroRNA����������������������������������������������������������������� 23, 316 Microsample proteomics��������������������������������������������������462 Morphogenesis��������������������������������������������������������� 368, 371 Mouse model����������������������������������������65, 69, 112, 115–116, 121, 252, 265, 266, 280, 314, 368, 371, 373, 378, 388, 392, 406, 410, 414, 416, 422, 423, 465, 493 Multiplex qPCR (m-qPCR)���������������������������� 193, 195, 197 Myeloperoxidase (MPO)���������������������������������� 485, 488, 493 N Natural transformation��������������������������������������������� 213, 237 Neutrophil extracellular traps (NETs)��������������������� 481–489, 492–496 Next generation DNA sequencing������������������� 128, 129, 217 NMR spectroscopy�������������������������������������������������������79–83 Nucleic acid techniques (for microbial taxonomy) broad-range PCR�������������������������������������������������������129 checkerboard DNA-DNA hybridization�������������������129 DNA-DNA hybridization������������������������������������������128 DNA microarray technology��������������������������������������129 fluorescence in situ hybridization (FISH)������������������128 multiplex PCR������������������������������������������������������������128 nested PCR����������������������������������������������������������������128 O Operational taxonomic unit (OTU)����������������� 155, 157–162 Oral diseases����������������������������������������������� 38, 132–135, 165 Oral fluids����������������������� 4, 37–39, 41–45, 47–50, 52–56, 58 Oral microbiota amplicon sequencing��������������������������������������������������156 taxonomy������������������������������������������������������������ 128, 168 Oral streptococci������������������������������������������������������ 204, 221 Organ culture dissection������������������������������������������������������������370–371 fixation����������������������������������������������������������������373–375 hanging drop culture����������������������������������� 369, 375–377 mouse����������������������������������������������������������������� 314, 368 Trowell-type organ culture����������������� 368, 369, 371, 378 Oxidative stress������������������������������������������������������������61–76 8-Oxo-2'-deoxyguanosine (8-OHdG)������������������������ 64–66, 69–70, 74 P PCR See Polymerase chain reaction PCR array������������������������������������������������� 301, 306, 450, 454 Periodontal diseases����������������������������������������� 37–39, 41–45, 47–50, 52–56, 58, 133–134, 309, 348, 350, 383 Periodontal ligament cell culture�������������������������������������������������� 309, 321, 407 fibroblasts�������������������������������������������������������������������413 processing����������������������������������������������������������� 415, 418 stem cells���������������������������������������������������� 414, 418–421 Periodontal pathogens���������������191–201, 314, 321, 381, 400 Polycaprolactone (PCL)������������403–405, 407, 408, 410, 411 Polymerase chain reaction quantitative real-time PCR����������������������������������������128 Standard PCR protocol����������������������������������������������397 Polymethyl methacrylate�������������������������� 171, 173, 180, 182 Porphyromonas gingivalis���������������������������130, 133, 191–195, 197, 198, 383, 390, 400, 491 Protein������������������������������������ 37–39, 49, 53, 55, 57, 58, 311, 315–317, 320, 462, 465–468, 471–473, 475, 477 analysis functional characterization��������������������������� 467, 473 identification�����������������������������������37, 38, 49, 53, 55, 57, 58, 466, 471–473, 475, 477 quantitation���������������������������������������������� 39, 58, 471 extraction��������������������������������������������311, 315–317, 320, 462, 465, 467–468, 477 purification����������������������������38, 300–302, 311, 315–317 Protein-releasing beads�����������������������������������������������������370 Proteomics microsample����������������� 461–463, 465, 467–473, 475, 476 Q qAMP������������������������������������������������������������������������������300 QIIME See Quantitative Insights Into Microbial Ecology qPCR See real-time quantitative PCR Qualitative and quantitative proteomics����������� 37–46, 48–58 Quantitative insights into microbial ecology������������153–162 Quantitative real-time reverse transcriptase PCR (qRT2-PCR)���������������������������������453–459 Oral Biology: Molecular Techniques and Applications 502 Index R Radiolabeling Of bacteria (3H-thymidine)����������������������������������������167 Of cultured cells, 310–311������������������������������������������314 Of yeast (35S-methionine)�����������������������������������167–169 Reactive oxygen species (ROS)������������62, 481–484, 486, 487 Real-time quantitative PCR (RT-qPCR)������������������ 17, 369, 372, 376 Recombinant proteins�������������������������������������� 188, 370, 372 Reduced representation bisulfite sequencing (RRBS)��������������������251–257, 261, 262, 279–294 Regional DNA methylation������������������������������������� 267, 269 RNA cRNA���������������������������������������������������������������������������17 extraction���������������������������������18, 34, 320, 323, 388, 395 gingival tissue�������������������������������������������������������������300 isolation������������������������������� 5, 9, 12, 18, 20–21, 385, 395 mRNA������������������������������������������������5, 17, 27, 263, 300, 349–360, 369, 385, 388–389, 395–397 Peripheral blood���������������������������������������������������������309 purification��������������������������������������������� 9, 300–302, 315 saliva����������������������������������������������������������� 5, 8–9, 17–35 RNA sequencing������������������������������������12, 17–35, 318–320, 324, 325, 337, 342 R software 2-sample T test�����������������������������������������������������������351 gplots������������������������������������������������������������������ 349, 357 S Saliva���������������������������������������������20, 49, 115, 396, 471, 473 collection human������������������������������������������������������������� 20, 115 mouse��������������������������������������������������� 112, 115–116 diagnostics���������������������������������������������������������� 3–12, 17 microarrays�������������������������������������������������������������������17 processing��������������������������������������������������5, 6, 10, 18, 20 proteomics 2-D gel electrophoresis�����������������������������������������471 Mascot database searching������������������������������������473 reverse transcriptase PCR (RT-PCR)�������������������396 SEQUEST database searching�������������������������������49 tandem mass spectrometry (LC-MS/MS)�������������48 Reverse transcriptase PCR (RT-PCR)�������������������23, 24 RNA isolation�������������������������������������������� 12, 18, 20–21 storage����������������������������������������������������������������������������4 submandibular������������������������������������ 115, 116, 120–121 transcriptome������������������������������������������������������������������5 Salivary gland���������������������������� 3, 5, 107, 108, 111–114, 121 Salivary hypofunction��������������������������������������� 108, 109, 113 Scanning electron microscopy (SEM)��������������������� 224, 236, 408, 409, 482, 485, 493, 496 Scintillation counter������������������167, 169–171, 180, 185, 188 SDS-PAGE See Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM See Scanning electron microscopy (SEM) Serum-free media (SFM)�����������������������������������������440–444 Signaling molecules���������������������������������� 369, 373, 375, 378 SigX-inducing peptide (XIP)���������������������������������� 219–225, 227–229, 234, 236, 238–239, 245 Silicone���������������������������������������������167, 171, 172, 179–180, 187, 188, 409 16S rDNA gene sequencing������������������������������������ 140, 141, 143–145, 153–162, 195 Small and long ncRNA profiling���������������������������� 18, 34, 35 Sodium dodecyl sulfate polyacrylamide gel electrophoresis��������������������������39, 45–49, 51, 53, 54, 57, 58, 174–175, 463–464, 468–471, 475, 476 Stable-isotope labeling chemistries�������������������������������50–54 Staphylococcus epidermidis���������������������������������� 166–168, 171, 172, 174, 179–183 adhesion to saliva-coated dentures������������������������������167 adhesion to saliva-coated medical-grade silicone����������������������������������������������������179–180 Statistical analysis������������������������������������������������������� 98, 264 Statistical model��������������������������������������������������������� 98, 260 Stem cells������������������������������������������������� 415–416, 419–420 cryopreservation���������������������������414, 420–421, 444, 451 differentiation�������������������������������������������������������������367 isolation adherence������������������������������������������������������419–420 immunomagnetic beads������������������������ 415–416, 419 mesenchymal����������������������� 414, 419, 421, 422, 439, 440 periodontal ligament����������������������������������� 403, 404, 407 Streptococcus/Streptococci S mitis������������������������� 130, 221–223, 225–227, 229, 230 S mutans����������������������������� 130, 133, 204, 207, 208, 215, 217, 219–226, 228–231, 233–235, 238–244 S pneumoniae��������������������������������������220, 233–235, 240, 241, 243, 245 Streptococcus mutans bacteriocin assays��������������������������������������������������������205 competence��������������������������220, 224, 229, 230, 234–235 mutagenesis����������������������������������������������������������������233 transformation������������������������������������220, 221, 223–225, 229, 230, 238–239 Streptococcus sanguinis Hydrogen peroxide production assay��������������������������204 SYBR green����������������������������������������������114, 118, 192, 269, 281, 284, 285, 305, 372, 376, 454 Synthetic peptides�������������������������������������������� 220–222, 234 Systems biology������������������������������������������������������������������79 T Tannerella forsythia���������������������������130, 133, 191–195, 198 TaqMan (probe)���������������������������������������������������������������192 Oral Biology: Molecular Techniques and Applications 503 Index Taxonomy������������������������������������������������� 128–135, 159, 160 Terminal restriction fragment length polymorphism��������128 DNA extraction��������������������������������������������������140–144 restriction enzymes��������������������������������������������� 140, 143 Tissue culture�������������������������������������������170, 177, 188, 370, 373, 385, 393, 415, 418, 441–443, 484 Tissue engineering�������������������������������������������� 403, 404, 414 Total RNA������������������������������������������22, 302, 312, 316–318, 320, 323, 376, 396, 448, 449, 452–454 Transcriptome�������������������������������������������� 12, 308, 309, 348 Transformation of anginosus group streptococci�������������������������� 227, 228 of Streptococcus mitis����������������������������������� 223, 225–227, 229, 230 of Streptococcus mutans�������������������������208, 220, 223–226, 228–230, 233, 234, 238–239, 244 of Streptococcus pneumoniae������������������ 220, 233, 234, 240 using synthetic CSPs������������������������������������������225–227 T-RFLP See Terminal restriction fragment length polymorphism V Viral vectors delivery������������������������������������������������������ 111, 115, 116, 120–121 recombinant serotype adenoviral (rAd5)��������� 108–110, 114–118, 122 serotype adeno-associated viral (rAAV2)�������� 108–111, 114–115, 118–120, 122 W Whole-genome bisulfite sequencing (WGBS)������� 251–255, 257, 261, 262, 280, 291, 292 X XIP See SigX-inducing peptide Z Zoledronic acid�����������������������������������������������������������������449 [...]... in a standardized, repeatable fashion and careful handling of the sample throughout the collection and downstream testing process This rule applies to all specimen types, but care should Gregory J Seymour et al (eds.), Oral Biology: Molecular Techniques and Applications, Methods in Molecular Biology, vol 1537, DOI 10.1007/978-1-4939-6685-1_1, © Springer Science+Business Media LLC 2017 3 4 Paul D. Slowey... (eds.), Oral Biology: Molecular Techniques and Applications, Methods in Molecular Biology, vol 1537, DOI 10.1007/978-1-4939-6685-1_2, © Springer Science+Business Media LLC 2017 17 18 Blanca Majem et al studies characterizing ncRNAs in saliva have used RNA-sequencing (RNA-Seq) technologies [13] In this chapter, we present the detailed methodology for RNA extraction, cDNA library construction and quality... markers in various malignancies [49], for noninvasive prenatal testing (NIPT, [50]), and for other diseases including rheumatoid disease, trauma, myocardial infarction, and fever and inflammatory disease [49, 51–54] Methods for the isolation of cfDNA again typically include blood, amniotic fluid, and other invasive bodily fluids While isolation of cfDNA has been carried out using saliva, the process involves... discovery of oral and systemic diseases Key words Saliva, exRNAs, Small and long ncRNA profiling, Biomarkers, RNA sequencing 1 Introduction Extracellular RNA (exRNA) in human saliva is an emerging field for noninvasive diagnostic applications The discovery of saliva-derived mRNA in normal and oral cancer patients [1–3] and other forensic applications [4, 5] opened up a new field for noninvasive molecular. .. syndrome in autism, disorders of the salivary glands, cancers (including breast, head and neck, and oral cancers), abused drug testing in the workplace and other environments, as well as certain systemic diseases including HIV, hepatitis C, and Sjögren’s syndrome The success of any test, whether for research or diagnostic purposes, relies on the successful harvesting of the specimen from a subject in a standardized,... raking/scraping tool that collects cells from the inside of the oral cavity (buccal mucosa) [23, 29] The collection head of the DNA⋅SAL™ tool is rubbed gently on the inside of the cheeks for 30 s, resulting in the accumulation of cells on the body of the DNA⋅SAL™ device In addition, cells are abraded by the mild raking action and remain “free-flowing” in the saliva in the pool formed in the mouth In. .. true in the case of marijuana, when testing for “impairment” and whether a particular individual is fit to drive a vehicle or perform dangerous tasks Multiple diseases have also been detected using saliva, including caries risk [12–14]; periodontitis [15]; oral [16], breast [17–22], and head and neck cancers [23]; and salivary gland disorders [24] Point of care tests are now also in development looking... from Illumina® platform for RNA sequencing Therefore, the chapter is divided into two main sections regarding the type of the library constructed (small and long ncRNA libraries), from saliva collection, RNA extraction and quantification to cDNA library generation and corresponding QCs Using these invaluable technical tools, one can identify thousands of ncRNA species in saliva These methods indicate... WS into each well (standard and samples) and incubate the plate for 15 min at RT in the dark (with lid) 9 Read the plate at 480–520 nm in a spectrophotometer (see Note 11) Agilent Bioanalyzer, Eukaryotic RNA Pico Chip 1 Take out the reagents 30 min prior to running the Chip and allow them to reach RT in the dark 2 Follow the manufacturer instructions for preparing the geldye-matrix properly, and. .. other applications, as has the QIAzol lysis reagent from the same company Other methods that have been used include organic extraction methods (TRIzol LS), spin filter-based methods (QIAamp Viral (Qiagen)), NucleoSpin (Clontech), and miRVana (Life Technologies) and combined method of organic extraction and spin filter clean up (miRNeasy micro (Qiagen)) and Quick-RNA MicroPrep (Zymo Research) 4 In reference ... much in continued research in the basic biological sciences This second edition of Oral Biology: Molecular Techniques and Applications continues the approach taken in the first edition and has... Seymour et al (eds.), Oral Biology: Molecular Techniques and Applications, Methods in Molecular Biology, vol 1537, DOI 10.1007/978-1-4939-6685-1_1, © Springer Science+Business Media LLC 2017 Paul D. Slowey... the profession lies in research, and it is anticipated that this second edition of Oral Biology: Molecular Techniques and Applications will continue to be a useful resource for oral biologists at