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Methods in Molecular Biology 1606 Virginia Espina Editor Molecular Profiling Methods and Protocols 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 Molecular Profiling Methods and Protocols Second Edition Edited by Virginia Espina Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA Editor Virginia Espina Center for Applied Proteomics and Molecular Medicine George Mason University Manassas, VA, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6989-0 ISBN 978-1-4939-6990-6 (eBook) DOI 10.1007/978-1-4939-6990-6 Library of Congress Control Number: 2017937315 © 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 The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 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 Dedication This book is proudly dedicated to Mary Anne and Len Schiff, for their initial faith in my future scientific career, and to my husband Tito; my children Ben, Paul, and Emily; and my favorite future scientist Olivia, for always listening to my ideas Virginia Espina v Preface The first edition of Molecular Profiling (published in 2012) was at the forefront of the personalized medicine movement The first edition included reviews of genomics and genomic profiling, technologies which in the intervening years have rapidly evolved into routine clinical assays for detecting mutations Mass spectrometry for protein profiling has also evolved into sensitive and specific multiple reaction monitoring assays, enabling quantification of proteins without antibody-based methods, while tumor staging and grading and tissue preservation continue to be important aspects of molecular profiling As you can see from these examples, staying current in molecular profiling requires lifelong learning and incorporating “routine” laboratory analyses with cutting-edge technologies Hence, when Professor John Walker, editor for the Methods in Molecular Biology series, inquired as to my interest in editing an updated version of this book, I was honored that the readers found the first edition informative and that there was sufficient, continuing interest in molecular profiling for an updated version However, I also knew that the second edition would require many updates to the protocol chapters to reflect the current state of the art in molecular profiling The purpose of this revised volume is to provide both an update on technology and an accelerated tutorial to assist students, entrepreneurs, new investigators, and established investigators who want to quickly become versed in, and immersed in, the entire process from discovery to clinical trial validation and commercial public benefit The aims of the first and second edition are the same: to span the full spectrum of molecular profiling from tumor staging and grading through biomarker discovery to commercialization The practical guides are not limited to experimental methods I have included tutorials on tumor staging, ethics, patents and intellectual property, product development, innovative clinical trial designs, and grant writing tips for investigators seeking funding in translational research Molecular Profiling, second edition, includes 17 new chapters and revised/updated chapters The new chapters cover some “tried-and-true” laboratory methods such as PCR and scanning electron microscopy The second edition also includes updated versions of antibody validation and Western blotting I had two reasons for including these “standard,” often kit-based, laboratory methods: (1) understanding the science behind the “kits” can help solve many problems encountered in assay development, and (2) the book is intended for a wide audience, including students and physician-scientists The 17 new chapters cover laboratory methods relevant to human disease: microsatellite analysis, somatic mutation analysis, proteomic bioinformatics, microscopic imaging, preservation of bone tissue for molecular profiling, glycomics, metabolomics, immunohistochemistry, FISH, ELISA development, and multiple reaction monitoring mass spectrometry Chapters 1–3 introduce the reader to clinical medicine through a primer on tumor staging and grading, ethics in medicine, and clinical trial design These chapters have been updated to address the current relevant information and issues For example, the chapter on clinical trials discusses examples of innovative trial design in which data generated during the clinical trial can be used to modify therapies administered to the patients as the trial is accruing patients vii viii Preface A set of core chapters (4–23) covering genomics, proteomics, imaging, and bioinformatics illustrate current laboratory protocols for generating data relevant to molecular medicine Each of these disciplines is complementary, and the grouping simply provides a means for differentiating the classes of molecular analytes An emphasis is placed on tissue- based molecular profiling, which is the core of personalized medicine Although many of the techniques discussed in this volume use commercially available reagents and instrumentation, it is imperative for the user/reader to understand the principles and nuances of these techniques, because they are designed for use with irreplaceable human tissue specimens The three topics covered in Chapters 24–26 are a unique aspect of this volume of the Methods in Molecular Biology series These latter chapters discuss, in a narrative or tutorial style, real-world needs in personalized molecular medicine The narrative chapters are designed to provide the reader with a well-rounded discussion of intellectual property issues in biotechnology, human subjects research requirements, tips for grant writing in translational research, and an overview of technology transfer (patent) issues As with the protocol chapters, important points are highlighted in the Notes section for each of the narrative chapters I hope that the readers of this second edition of Molecular Profiling will use it as a practical guide at the lab bench as well as in the classroom The intended readership spans the range of scientists, pathologists, oncologists, residents, biotechnologists, medical students, and nurses involved in clinical trial research I would like to express my sincere gratitude to my editorial assistant, Emily Espina, who provided excellent grammar editing I truly appreciate, and thank, all my authors for their time and effort in compiling and submitting new and updated chapters Their collective contributions and input have greatly expanded the scope and depth of the book I thank Lance Liotta, my co-editor on the first edition, who supported me with the utmost respect and trust, while I pursued this solo editing endeavor I anticipate that this revised volume will attract new investigators, and invigorate experienced researchers, who can apply their creative talents to realize the promise of individualized molecular medicine I hope you find this revised edition a useful and informative guide for your molecular profiling adventures Manassas, VA, USA Virginia Espina Contents Preface vii Contributors xi Tumor Staging and Grading: A Primer Stacy M Telloni Innovations in Clinical Trial Design in the Era of Molecular Profiling Julia D Wulfkuhle, Alexander Spira, Kirsten H Edmiston, and Emanuel F Petricoin III Personalized Medicine: Ethical Aspects G Terry Sharrer Antibody Validation by Western Blotting Michele Signore, Valeria Manganelli, and Alex Hodge Scanning Electron Microscopy Sample Preparation and Imaging Jenny Ngoc Tran Nguyen and Amanda M Harbison One-Step Preservation and Decalcification of Bony Tissue for Molecular Profiling Claudius Mueller, Michael G Harpole, and Virginia Espina Application of Hydrogel Nanoparticles for the Capture, Concentration, and Preservation of Low-Abundance Biomarkers Ruben Magni and Alessandra Luchini Using Laser Capture Microdissection to Isolate Cortical Laminae in Nonhuman Primate Brain Brian A Corgiat and Claudius Mueller Western Blot Techniques Brianna Kim 10 ELISA for Monitoring Nerve Growth Factor Justin B Davis 11 Reverse Phase Protein Microarrays Elisa Baldelli, Valerie Calvert, Alex Hodge, Amy VanMeter, Emanuel F Petricoin III, and Mariaelena Pierobon 12 Clustering and Network Analysis of Reverse Phase Protein Array Data Adam Byron 13 PCR: Identification of Genetic Polymorphisms Amanda M Harbison and Jenny Ngoc Tran Nguyen 14 Microsatellite Analysis for Identification of Individuals Using Bone from the Extinct Steller’s Sea Cow (Hydrodamalis gigas) Jeffery F Warner, Michael G Harpole, and Lorelei D Crerar ix 19 37 51 71 85 103 115 133 141 149 171 193 205 x Contents 15 Somatic DNA Mutation Analysis Anthony O’Grady and Robert Cummins 16 Optimization of Immunostaining for Prospective Image Analysis Scott M Lawrence and Yelena G Golubeva 17 Fluorescence In Situ Hybridization of Cells, Chromosomes, and Formalin-Fixed Paraffin-Embedded Tissues Ahmad Alamri, Jun Yeb Nam, and Jan K Blancato 18 High-Resolution Image Stitching as a Tool to Assess Tissue-Level Protein Distribution and Localization Bryan A Millis and Matthew J Tyska 19 Mass Spectrometry-Based Biomarker Discovery Weidong Zhou, Emanuel F Petricoin III, and Caterina Longo 20 Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM) Paul Russo, Brian L Hood, Nicholas W Bateman, and Thomas P Conrads 21 LC-Mass Spectrometry for Metabolomics Allyson L Dailey 22 Metabolomic Bioinformatic Analysis Allyson L Dailey 23 Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry Stefan Mittermayr, Simone Albrecht, Csaba Váradi, Silvia Millán-Martín, and Jonathan Bones 24 Grant Writing Tips for Translational Research Lindsay Wescott, Michael Laskofski, Donna Senator, and Carly Curran 25 Inventions and Patents: A Practical Tutorial Hina Mehta, Lille Tidwell, and Lance A Liotta 26 Product Development and Commercialization of Diagnostic or Life Science Products for Scientists and Researchers Meghan M Alonso 219 235 265 281 297 313 333 341 353 367 379 399 Index 409 Contributors Ahmad Alamri • Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA; Department of Clinical Laboratories Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia Simone Albrecht • NIBRT—The National Institute for Bioprocessing Research & Training, Dublin, Ireland Meghan M. Alonso • IMUA Services, Medical Invention and Device Development Consulting, Carlsbad, CA, USA Elisa Baldelli • Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA Nicholas W. Bateman • DOD Gynecologic Cancer Center of Excellence, Annandale, VA, USA Jan K. Blancato • Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA; Georgetown Lombardi Comprehensive Cancer Center, Fisher Center for Hereditary Cancer and Clinical Genomics Research, Georgetown University, Washington, DC, USA Jonathan Bones • NIBRT—The National Institute for Bioprocessing Research & Training, Dublin, Ireland Adam Byron • Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Valerie Calvert • Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA Thomas P. Conrads • Inova Dwight and Martha Schar Cancer Institute, Falls Church, VA, USA; Gynecologic Cancer Center of Excellence, Women’s Health Integrated Research Center, Annandale, VA, USA Brian A. Corgiat • Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA Lorelei D. Crerar • Department of Biology, George Mason University, Fairfax, VA, USA Robert Cummins • Department of Pathology, RCSI Education & Research Center, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland Carly Curran • Office of Sponsored Programs, George Mason University, Fairfax, VA, USA Allyson L Dailey • Department of Chemistry and Biochemistry, George Mason University, Manassas, VA, USA Justin B. Davis • Department of Chemistry and Biochemistry, George Mason University, Manassas, VA, USA Kirsten H. Edmiston • Departement of Surgery, Inova Fairfax Hospital Cancer Center, Falls Church, VA, USA Virginia Espina • Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA Yelena Golubeva • Medimmune, Gaithersburg, MD, USA Amanda M. Harbison • Northern Virginia Community College, Manassas, VA, USA xi Chapter 26 Product Development and Commercialization of Diagnostic or Life Science Products for Scientists and Researchers Meghan M. Alonso Abstract Commercializing a diagnostic or life science product often encompasses different goals than that of research and grant funding There are several necessary steps, and a strategy needs to be well defined in order to be successful Product development requires input from and between various groups within a company and, for academia, outside entities The product development stakeholder groups/entities are research, marketing, development, regulatory, manufacturing, clinical, safety/efficacy, and quality After initial research and development, much of the work in product development can be outsourced or jointly created using public–private partnerships This chapter serves as an overview of the product development process and provides a guide to best define a product strategy Key words Commercialization, Design for manufacturing, Diagnostic, Go-to-market strategy, Product development, Public–private partnerships, Scale-up 1 Introduction Product development and commercialization can often be intimidating and overwhelming to the scientist or researcher that has typically worked in research based on competitive grant funding systems with experimental milestones and goals Product development and commercialization require discussions with many stakeholders, at the earliest stages of product development At the pilot stage of development, discussions should be started simultaneously with research, marketing, and regulatory groups to determine the initial feasibility of product development [1] Further discussions with development, manufacturing, clinical, safety/efficacy, and quality stakeholders are also essential elements of product development Much of this work can be outsourced or jointly developed through public–private partnerships [2] (see Note 1) Market research and fund-raising are the first of many challenging steps in the process Virginia Espina (ed.), Molecular Profiling: Methods and Protocols, Methods in Molecular Biology, vol 1606, DOI 10.1007/978-1-4939-6990-6_26, © Springer Science+Business Media LLC 2017 399 400 Meghan M. Alonso Financing product development is of course a major hurdle for most life science researchers whose ideas/products are funded through competitive grants Many governmentally funded grants are awarded based on novel hypotheses that have a potential substantial impact However the available budgets are usually designated to perform research to prove the hypothesis and not include product development costs [2] Technology Transfer Offices, public–private partnerships, and not-for-profit development partnerships can potentially provide the gap funding needed to sustain product development and commercialization or assist in evaluating whether or not a new product will be adopted into national guidelines and practices [2–5] The most important strategy in terms of potential product success is establishing how the product will be positioned in the marketplace and where it will be marketed Unlike most grant-funded research, the goal of product development is commercialization The commercialization strategy is prioritized and determined from market research regarding the best ways to leverage the intellectual property around the product [6, 7] The product development process is then mapped out with design for manufacturing at the forefront to meet the marketing and technical requirements for the end user It is extremely helpful to identify manufacturing partners, if manufacturing will be done on a contract basis, as early on as possible One must weigh the options of onshore or offshore manufacturing so these processes can be defined in development This chapter outlines the required steps for product development as well as a framework for identifying an overall commercialization strategy 2 Materials 2.1 Intellectual Property and Strategy Intellectual property rights are created as part of national law [8] International treaties strive to synchronize intellectual property owners’ rights, but the treaties vary by country [8] However the complexities of intellectual property and international treaties require interpretation by intellectual property attorneys But how you know which intellectual property attorney to choose? Three principles should guide your decision: Their track record: Find out how many patents they have successfully filed and the nature of the patent, e.g., a utility patent or a design patent (see Note 2) Your product’s landscape: You need an attorney that is familiar with your product’s landscape, i.e., the industry and scientific background/technology The attorney should also be knowledgeable regarding the currently issued patents and patent applications in your product domain Commercialization of Diagnostic Products 401 Freedom to operate: Your attorney will need to conduct thorough research on the patent landscape to ensure you have freedom to operate if there are any existing patents around your technology The intellectual property attorney should be able to identify ways you can work without infringing on the existing patents Oftentimes, intellectual property will be created during product development Therefore identification of your product’s landscape and the freedom to operate are done in tandem with product development Once the intellectual property is well defined and there is evidence of freedom to operate, a strategy regarding the future of the product’s intellectual property needs to be in place Strategies regarding product intellectual property include: Leveraging IP to fully develop the product and take it to market Aiming the intellectual property and product for technology licensing Developing the product to a point in which it can be acquired by the competition or a large corporation that sees value in your technology for their customer’s needs 2.2 Market Research While the attorney is working on patent research, to maximize time, market research can be started Market research is the most aspect of product development and should be done at the prototype stage Market research ensures that the product meets all expectations for the developer, the end user, influencers, and regulatory requirements [1] Pharmaceutical companies often use a ‘target product profile” that defines the product attributes and delineates the minimally acceptable requirements of the product from its desired attributes [1] (see Note 3) As a baseline for market research, about 20 interviews with stakeholders, including potential users, purchasers, influencers, and critical partners, should be conducted Formulate a set of questions about your area of expertise to ask each group Example initial market research questions: A question to ask potential users would be: What is your current workflow and what would make this easier? For a purchaser: Why you buy this particular product and what influences your purchasing decision? For an influencer: What gets you most excited about new or disruptive products such that you would want to share it with others? For the critical partners: What technologies are you focusing on acquiring or licensing and how can my product help you? 402 Meghan M. Alonso Next, take inventory of the feedback you have received and analyze how you can meet your stakeholders’ needs, wants, and desires Once that process is established, preliminary research can be expanded with more targeted questions, ideally, to around 150 interviewee participants After it has been determined from preliminary market research that the market does in fact demand the product in question, formal market research should be started by engaging with a company or consultant The information gathered in this more formal research should prove or disprove viability in the marketplace, and the decision to move forward with product development can be determined at this point [7] 2.3 Reimbursements (If Applicable) If the product in development will rely on a reimbursement from insurance companies as payment, the research on this needs to be started early on and could be started in parallel with market research [3–5] The reimbursement research provides critical information that shapes the later stages of market research Researching reimbursement rates can be done by calling the insurance companies, but given that there are several plans in several states (for the US market), it is helpful to hire a company, or a consultant, that is knowledgeable and has expertise in your products in vitro device category, sample preparation, or life sciences area and/or the application (such as oncology diagnostics, rapid detection of viruses, software algorithms, etc.) The prerequisites for analyzing reimbursement rates should be well thought out prior to commencing a study: Geographical markets: The geographical region for the eventual product sale and the region for analyzing possible reimbursement should be similar Product application: Select the optimal market for each product application There may be more than one application, and one should be chosen as far as the best market However, when researching reimbursements, all product applications can be researched to determine the best path toward commercialization and the ability to have insurance company reimbursement for the product If one path has an extremely low reimbursement rate but the other path has a high rate, it may make sense to move forward with the product application that has the higher rate The caveat is that the end-user market will most likely change, thus affecting the initial market research Current reimbursement landscape: Determine the reimbursement rate for similar or competitive products Value of the new product: Sometimes, seeking a higher reimbursement rate than the competition can be a great strategy For example, if a new product is a molecular diagnostic that Commercialization of Diagnostic Products 403 replaces a 48 h test with a 3 h test and can reduce the length of the hospital stay as well as the cost of antibiotics, insurance companies will most likely favor this product and pay the additional cost for the higher reimbursement rate since funds are saved elsewhere 2.4 Regulatory (If Applicable) If your product requires government regulation, it is best to initiate the regulatory plan once market research is complete and the product positioning in the market is determined [9] For any market, an intended use statement must be drafted along with instructions for use [9–11] Depending on the country of choice for distribution, there will be different regulatory requirements so consultation with a specialist in the product space along with the knowledge of the desired country is highly recommended Establish a regulatory plan Confer with a specialized regulatory consultant or company that has expertise in your product’s particular area to ensure a proper pathway for your product Start with an intended use statement The intended use statement provides an overview of how the product will be used, who will be using it, and for what it will be used Develop instructions for use Specific guidelines need to be followed for the “instructions for use.” With your consultant or hired company to develop this document Prepare plans for future design, development, and scale-up: Regulatory agencies can provide valuable feedback and prevent costly reworking of products if they are consulted often and early in the product development process However, assuming that the product regulatory application is successful, valuable time can be saved if the design, development, and scale-up are being planned while working on regulatory submission materials 3 Methods 3.1 Engineering/ Assay Development for Manufacturing If your product has a device component to it (e.g., lateral flow cartridge, molecular diagnostic system, visual or fluorescence reader, lab automation, or sample prep), the device will need prototyping for proof of concept and then will need to be designed for manufacturing and scaled up according to anticipated annual volumes [12] This same process will need to be completed for assay development as well [13] Many times, a technology will work well on the bench but when trying to commercialize and replicate becomes extremely difficult A key aspect for manufacturing is to have an experienced, diversified team in the field of diagnostics, life sciences, and/or sample preparation working on the scale-up of your product that 404 Meghan M. Alonso are up-to-date with the latest technology, reagents, material sciences, assembly methods, and a thorough understanding of the manufacturing process in order to properly build the product This also helps with speed to market Sometimes, companies sacrifice talent by choosing to work with a low-priced company but then later realize that the lower-priced company takes two to three times as long to develop the product than the more expensive company Usually, the more expensive companies charge a premium because of their knowledge and experience, which enables them to develop the product faster than other companies Companies should also consider the diverse team approach Most of the time, diverse teams work better than individuals since teams bring several different perspectives together to solve problems In the engineering and assay development sequence of events, the following steps are: Product requirements are gathered and organized from marketing, technical, and regulatory departments, consultants, or hired companies All of these requirements shape the design and scope of the project in order to provide an accurate timeline and costs A feasibility study/phase may be applicable Concepts for a scalable design are produced, tested, and refined Iterations on the selected concept are completed, establishing proof of concept Packaging engineering and studies commence Design scale-up processes are launched Design incorporates feedback from formative usability study and preclinical study Manufacturing and assembly processes are established Design is transferred to manufacturing 3.2 Usability Studies Once again, if the product will be marketed to a regulated industry, a regulatory plan, along with usability and clinical studies, will need to be completed [13, 14] Depending on which markets are selected for penetration, different standards are required In the US market, the Food and Drug administration (FDA) requires usability and human factor studies to show that users can properly interact with the device, it is intuitive, and no one will get hurt or misuse the device as a result of the design and instructions The FDA highly encourages a dialogue as early as possible, and the use of formative studies can be extremely helpful These are studies done while the product is still in development and allows the feedback from the FDA to shape the remaining design and development of the product, therefore, increasing the chances that it will be Commercialization of Diagnostic Products 405 approved once development is complete As mentioned, the next step in usability studies is the summative studies These usually take place once the design is established and fixed and the product is heading into clinical studies The summative studies are the final step toward approval from a usability standpoint with the FDA If a company skips the formative studies and heads straight into the summative studies, they are putting themselves at risk for a repeat of the design work if the FDA reports findings that are not suitable for customer interaction Engage the regulatory agency early and often and allow the regulatory agency to partake in the formative studies 3.3 Clinical Studies Clinical studies include the design of observations and experiments to prove efficacy, safety, reliability, and repeatability of your product [10, 11, 13, 14] The established regulatory plan will shape the plan for clinical studies A preclinical study can be conducted to gather data as the product is in development similar to that of a formative usability study The preclinical studies provide for early engagement with the FDA (or other regulatory body) and data to help shape the design of the product When choosing a team to oversee clinical studies, it is important to note the following: Each study, depending on the complexity, could require a different number of participants Depending on the device or assay, there could be more than one study needed For example, one study for specimen collection and another for the diagnostic component would be needed to satisfy clinical requirements Participants must be diverse Clinical studies are conducted over several sites across the country to test various demographics, socioeconomic levels, and regional norms 3.4 Transfer to Manufacturing As mentioned above, a product should be designed for scalability, working with the end user and market niche in mind The product development should be agnostic in that no manufacturing method should be excluded when determining how to scale-up Once limitations are set, development is hindered, and solutions to pressing problems are not formulated as fast Early engagement with a contract manufacturer (or in-house manufacturing team if the product will not be outsourced) ensures a smooth transition Some important considerations for manufacturing are: Determine distribution and sales channels early on This will have an influence on where manufacturing takes place If a manufacturer is selected in China but only small volumes are needed in the USA that don not have a long shelf life, China may not be the best choice due to logistical challenges 406 Meghan M. Alonso Development team and manufacturer need to work together on manufacturing and assembly processes to help ease transition and speed time to market Source a reliable choice(s) of materials for the product so if one is out of stock, the other is validated and ready to be placed in production 4 Notes Companies and/or consultants can provide very valuable input and resources for product development, particularly with commercialization as a goal Most of the covered topics can be outsourced, and http://imua-services.com and http://toolboxmed.com are great places to start teaming up with development partners The National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health has funding initiatives for translational research https://ncats.nih gov/funding A utility patent is issued for the inventions of a new and useful process, machine, manufacture, or composition of matter A design patent is issued for a new, original ornamental design embodied in or applied to an article of manufacture (Source: United States Patent and Trademark Office, www.uspto.gov) Specific product requirements should be addressed in the target product profile For a pharmaceutical product, these attributes may be the route of administration, packaging, compatibility with intravenous delivery devices, and type of release (sustained release or immediate) [1] References Lambert WJ (2010) Considerations in developing a target product profile for parenteral pharmaceutical products AAPS PharmSciTech 11(3):1476–1481 doi:10.1208/ s12249-010-9521-x Rose LM, Everts M, Heller C, Burke C, Hafer N, Steele S (2014) Academic medical product development: an emerging alliance of technology transfer organizations and the CTSA. Clin Transl Sci 7(6):456–464 doi:10.1111/ cts.12175 Bishai DM, Champion C, Steele ME, Thompson L (2011) Product development partnerships hit their stride: lessons from developing a meningitis vaccine for Africa Health Aff (Millwood) 30(6):1058–1064 doi:10.1377/hlthaff.2011.0295 Mahoney RT (2011) Product development partnerships: case studies of a new mechanism for health technology innovation Health Res Policy Syst 9:33 doi:10.1186/1478-4505-9-33 Wells WA, Brooks A (2011) Adoption of new health products in low and middle income settings: how product development partnerships can support country decision making Health Res Policy Syst 9:15 doi:10.1186/1478-4505-9-15 Jahn R, Muller O, Bozorgmehr K (2015) Characteristics and determinants of knowledge transfer policies at universities and public institutions in medical research—protocol for a systematic review of the qualitative research literature Syst Rev 4:110 doi:10.1186/ s13643-015-0094-3 Jekunen A (2014) Decision-making in product portfolios of pharmaceutical research and development—managing streams of innovation in Commercialization of Diagnostic Products highly regulated markets Drug Des Devel Ther 8:2009–2016 doi:10.2147/DDDT.S68579 Carroll MW (2015) Sharing research data and intellectual property law: a primer PLoS Biol 13(8):e1002235 doi:10.1371/journal pbio.1002235 Pramod K, Tahir MA, Charoo NA, Ansari SH, Ali J (2016) Pharmaceutical product development: a quality by design approach Int J Pharm Investig 6(3):129–138 doi:10.4103/2230-973X.187350 10 Hakemeyer C, McKnight N, St John R, Meier S, Trexler-Schmidt M, Kelley B, Zettl F, Puskeiler R, Kleinjans A, Lim F, Wurth C (2016) Process characterization and design space definition Biologicals 44(5):306–318 doi:10.1016/j.biologicals.2016.06.004 11 Ohage E, Iverson R, Krummen L, Taticek R, Vega M (2016) QbD implementation and Post Approval Lifecycle Management (PALM) Biologicals 44(5):332–340 doi:10.1016/j biologicals.2016.06.007 407 12 Jain A, Taghavian O, Vallejo D, Dotsey E, Schwartz D, Bell FG, Greef C, Davies DH, Grudzien J, Lee AP, Felgner PL, Liang L (2016) Evaluation of quantum dot immunofluorescence and a digital CMOS imaging system as an alternative to conventional organic fluorescence dyes and laser scanning for quantifying protein microarrays Proteomics 16(8):1271– 1279 doi:10.1002/pmic.201500375 13 Gascon P, Fuhr U, Sorgel F, KinzigSchippers M, Makhson A, Balser S, Einmahl S, Muenzberg M (2010) Development of a new G-CSF product based on biosimilarity assessment Ann Oncol 21(7):1419–1429 doi:10.1093/annonc/mdp574 14 Thomas JA, Collins CR, Das S, Hackett F, Graindorge A, Bell D, Deu E, Blackman MJ (2016) Development and application of a simple plaque assay for the human malaria parasite plasmodium falciparum PLoS One 11(6):e0157873 doi:10.1371/journal pone.0157873 Index A Adenocarcinomas�� 4–8, 10, 13, 34 Agarose��194, 196, 198, 199, 201, 202, 208, 211–212 Alkaline phosphatase�� 56, 57, 62–64, 68 Alkylate��304, 318, 322, 324, 327 America Invents Act��384 Amplification�� 69, 87, 150, 152, 160, 162, 175, 194, 195, 200, 209, 220, 228, 243, 244, 246, 254, 257, 276 Antibody antibodies�� 51–54, 62, 64, 66, 68, 135, 141, 143, 145, 149, 152, 161, 167, 171, 176, 180–181, 186, 238, 244, 254, 257, 258, 306, 361, 383 capture antibody��142–145 primary antibody�� 55, 56, 59, 61–64, 67–69, 135, 137, 139, 142, 152, 160–164, 167, 238, 243–247, 252–254, 256–258 secondary antibody��53, 55–57, 59, 62–64, 67–69, 136, 137, 142, 152, 161–163, 167, 246, 248, 253, 254, 257, 259 Antigen unmasking�� 245, 256 Ataxia telangiectasia mutated (ATM)�� 318, 319 Avidin-HRP��142–146 B Basement membrane��3 Bayesian adaptive design��30 Bioinformatics�� 297, 341–351 Biomarkers�� 4, 20, 22, 26, 28, 30–33, 40, 51, 85, 86, 103–111, 150, 219, 235, 238, 243, 247, 249, 251–257, 260, 297–310, 319, 326, 342, 343, 367, 368 Biotinyl tyramide��150 BLAST�� 195, 315 Body fluids�� 47, 55, 103, 104, 107, 109, 151, 193, 297, 304 Bond Breaker�� 55, 118, 126, 136 Bone�� 2, 4, 7, 10, 23, 87, 88, 90, 97, 205–212, 214–216 Bony tissue osseous tissues��88–89 Borrelia burgdorferi�� 133, 135 Breast cancer cribriform��8, 11 DCIS��9–11 inflammatory carcinoma��11 invasive breast carcinoma��11, 12 Buccal cell See Cheek cell Budget�� 371, 373–374, 400 C CA-125��12 Calcium�� 56, 57, 88, 152, 160, 214, 269 Cancer�� 1–15, 19–26, 28, 29, 31, 33, 38, 39, 45, 72, 87, 103, 176, 195, 265, 276, 382, 390 Capillary electrophoresis (CE)�� 207, 212, 213, 216, 353, 354, 356, 361, 363–365 CapSure�� 118, 122, 126, 127, 129, 164 Carry-over�� 67, 120, 166, 222, 232, 233, 254, 328 Cell culture�� 141, 144, 150, 151, 153, 199, 265, 266, 269–270, 274–276, 278, 316, 317, 322 Cell lysates�� 54, 55, 57, 152, 154, 156, 157, 176, 306, 316, 317 Cell lysis buffer��153 Cell signaling�� 54, 172 Cheek cells See Buccal cells Chelex resin�� 195, 197, 198, 200 Chemiluminescence�� 62, 63, 68, 135 Chromosome spreads��269–270 Clinical studies��235, 374–376, 404, 405 Clinical trial adjuvant trial�� 20, 25, 26 APHINITY trial��26 basket trial��27–30 CLEOPATRA��25 CUSTOM trial��28–30 gene therapy trial��44, 45 informed consent�� 41–42, 45, 47, 48 I-SPY trial�� 25, 31–33 NCI-MATCH trial��28, 29 NeoSphere trial��26 platform trial�� 27, 30–33 TAPUR trial��28, 29 trial model��20 umbrella trial��27 Cluster 3.0�� 175–178, 182, 183 Coinventors��383–384 Collision-induced dissociation (CID)�� 308, 309, 313–315, 319, 321, 324, 325, 327, 328 Commercialization�� 379, 399 Virginia Espina (ed.), Molecular Profiling: Methods and Protocols, Methods in Molecular Biology, vol 1606, DOI 10.1007/978-1-4939-6990-6, © Springer Science+Business Media LLC 2017 409 Molecular Profiling: Methods and Protocols 410 Index Computational analysis��173 Conditioned medium��144 Confidential disclosure agreement (CDA)�� 385, 395 Correlation network�� 342, 343, 348, 351 Cryopreservation solution OCT�� 118, 119 Cytoscape�� 175, 178, 179, 181, 182, 184–187 Ethylenediaminetetraacetic acid (EDTA)�� 88–90, 208, 210, 214 Extracellular matrix��3 Extraction buffer�� 55, 57, 65, 117, 118, 126, 127, 131, 151, 152, 154–157, 165, 224, 301, 304 D Feasibility�� 399, 404 Fixation fixative solution�� 153, 163, 300, 303 TheraLin�� 86, 89–92, 94, 96–100 Fluorescence FLR detector�� 356, 357 Fluorescence in situ hybridization (FISH)��265–279 Food and Drug Administration (FDA)�� 22, 23, 25–26, 28, 29, 33, 45, 404, 405 Formalin fixed paraffin embedded (FFPE)�� 127, 220, 221, 223, 231, 232, 241, 244, 252, 256, 265–279 DAPI�� 238, 245, 247–249, 253, 254, 257, 258, 260, 268, 270, 274–276, 279 Data analysis�� 153, 161, 163–164, 177, 230, 341–343 DCIS See Breast cancer Decalcification��85–100 Demineralization See Decalcification Dendrogram��173, 176, 178, 183, 184, 342, 343, 348 Deparaffinized��95 Desalting��299, 301, 305, 316, 318, 324, 325, 364 Diagnostic�� 20, 33, 40, 46–48, 52, 107, 150, 219, 220, 231, 281, 368, 379–381, 389, 390, 399 Diethyl pyrocarbonate (DEPC)�� 98, 196 Differentiation�� 3, 6, Digested�� 298, 299, 301, 314, 315, 318, 320, 322, 324, 327, 362 Digital PCR (dPCR)��194 Dimethyl sulfoxide (DMSO)�� 97, 196, 355, 362 Dithiothreitol (DTT)��300, 301, 304, 316, 318, 322, 354, 364 DNA dsDNA��194 mtDNA��206 nuclear�� 205, 206 synthesis��194 DNase�� 98, 195, 196, 200 Docetaxel��25 Dye-labeled dideoxynucleotides (ddNTPs)��212 E Electropherogram�� 354, 359, 361, 365 Electrophoresis Coomassie�� 151, 154, 299, 300, 302–303 1-D gel electrophoresis�� 55–56, 65, 298 silver staining�� 299, 300, 302, 303 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)�� 55, 65, 136–137, 299, 300, 302–303 Endoglycosidase digestion buffer��354 Enzyme-linked immunosorbent assay (ELISA) sandwich ELISA��141 Epidermal growth factor receptor (EGFR)�� 4, 15, 28 Erlotinib��28 F G Gas chromatography-mass spectrometry (GC-MS)�� 333, 334 Germline mutations��219 Gleason pattern��8 Global metabolomics��335 Glycan analysis glycans�� 353–355, 357–365 glycoproteins�� 12, 354, 357, 361–362, 364 glycosylation�� 361, 362, 364 Grant writing��367–377 H Health Insurance Portability and Accountability Act (HIPAA) patient privacy��43–44 Hematoxylin and eosin (H&E)�� 4, 21, 24, 91, 128, 221–224, 231, 241, 252, 267, 270–272, 277 Heterogeneity�� 115, 231 Hierarchical clustering hierarchical cluster analysis��175–178 High-performance liquid chromatography (HPLC) nano-HPLC��316, 317, 320–321, 323–325 ultra low pressure chromatography (ULPC)��335, 339 ultra performance liquid chromatography��316 Histology��3–8, 10–15, 29, 33, 34, 86, 89, 221 Histomorphology��86, 87, 97, 117, 277 Horseradish peroxidase (HRP)��56, 57, 63, 64, 68, 135, 141, 142, 150, 246 Hybridization�� 96, 265–279 Hydrophilic interaction liquid chromatography (HILIC)�� 356, 358, 359, 361, 362, 364, 365 I I-Block��56, 60–62, 68, 138, 139 Imaging image acquisition��163–164, 236, 239, 244, 247–250, 257, 284, 292 image analysis��156, 163, 167, 235, 237–250, 252–254, 256–260 ImageJ�� 284, 289 Immunoassay�� 104, 105, 107, 109 Immunoblotting See Western blot Immunocytochemistry��176 Immunofluorescence (IF)��235, 238, 243–249, 252–254, 256, 259, 261 Immunohistochemistry (IHC)��4, 33, 51, 89, 91, 176 Immunostaining��4, 54, 150, 152, 153, 159–162, 166, 235–250, 252–260, 283, 292 Individualized therapy�� 20, 380 Infra-red (infrared) IRDye680 Streptavidin�� 152, 160–162 In-gel digestion�� 299–301, 303–304 In situ hybridization (ISH)�� 88, 89, 96, 98 Institutional Animal Care and Use Committee (IACUC)�� 239, 372 institutional Review Board (IRB)��47, 48, 368, 371–372 Intellectual property��380, 382, 392, 396, 400–401 Interaction networks interaction network analysis�� 172, 174, 175, 178–182, 184, 185 Inventions�� 379–396, 406 Iodoacetamide��300, 301, 304, 309, 316, 318, 322, 354 Ionization��299, 307, 314, 334, 353, 354, 356, 357 Isoforms�� 52, 55, 64, 141 Isotonic salt solution��195 J Java TreeView�� 175, 176, 178, 184 Just in Time��371 L Laemmli sample buffer��136 Laser laser scanner�� 153, 163, 164 Laser capture microdissection (LCM) ArcturusXT��65 IR capture�� 116, 118–127 IR spot�� 123, 124 laser spot size�� 121, 129, 130 LCM cap��116–118, 121–123, 126, 127, 129, 130, 154, 155, 164, 165 PixCell IIe��65 Laws of nature��383 Molecular Profiling: Methods and Protocols 411 Index Liquid chromatography-mass spectrometry (LC-MS)��302, 304, 305, 327, 333–339, 354, 356, 359, 361 Loading dye��196, 198, 202, 208, 212 Lower limit of detection (LLOD)�� 318, 324–326, 329, 330 Lower limit of quantitation (LLOQ)�� 315, 325 Lung cancer�� 3–6, 15, 22, 28 Lyme disease�� 133, 134 Lymph node lymphatics��9, 13 sentinel lymph node (SLN)��9, 10 Lysis buffer�� 151, 153, 154, 208, 216, 325 M Macrodissection�� 221–225, 231 Manufacturing��383, 399, 400, 403–406 Market research��399–403 MassARRAY�� 220, 222, 225–227, 229–230, 232 Mass spectrometry��313 electrospray�� 195, 299, 314 isotope dilution��313–330 isotopic tags��362 mass spectrometer��225, 297, 308, 314, 316, 317, 320, 321, 326, 327, 329, 333, 335 mass spectroscopy��40 matrix assisted laser desorption time of flight (MALDI)��20 multiple reaction monitoring (MRM) selected reaction monitoring (SRM)��313 m/z�� 315, 319, 320, 327–329, 356, 357, 360 QTOF�� 335, 337, 356, 361 SEQUEST��299, 302, 309, 317, 320 Skyline�� 317, 324, 325, 330 stable isotope standard (SIS peptide)�� 315, 317, 319, 322–324 Master Mix�� 196, 197, 200, 211, 215, 227, 228 Medical ethics��44 Belmont Report��43, 44 eugenics�� 39, 41–42 informed consent Jesse Gelsinger��44 Nuremberg Code��42–44 Tuskegee Syphilis Study��42, 43 Melting temperature��195 Metabolomic fecal��335 feces��335 metabolites��107, 108, 174, 333, 335, 341–343, 348, 349 pheatmap�� 343, 348 qgraph�� 343, 348 Metastasis�� 1–3, 8, 10, 14, 24, 27, 87 Molecular Profiling: Methods and Protocols 412 Index Microsatellite loci dugongs��206 manatee�� 206, 207, 215 Steller’s sea cows��207 Microscope�� 72, 76, 116, 118, 119, 121–123, 125, 165, 221, 222, 224, 238, 267, 269–271, 274, 276, 277, 282–284, 291–294 Microscopy�� 4, 71–83 Microtome��90, 91, 98, 221, 222, 238, 242, 251, 267, 270 Modeling�� 185, 343 Moderately differentiated��3 Molecular profiling��19–34, 73, 85–87, 91–93, 97, 150, 193–195, 235, 281, 283, 319, 380 Molecular weight�� 52–55, 57, 58, 64–69, 104, 135, 136, 196, 198, 199, 334 Multi-dimensional microscopy stitching�� 281–287, 290–295 super-resolution��282 Multi-omic��33 Multiple myeloma�� 28, 389 Multiplex IF��238, 243, 244, 247–249, 253, 254, 256, 261 Multispectral imaging�� 253, 256 N Nanoparticles hydrogel�� 103, 105 Nanotrap�� 133, 135 vinylsulfonic acid (VSA)��104 Nanowires��73 National Institutes of Health (NIH)�� 45, 368, 369, 371–373, 376, 377 Necrosis�� 6, 8, 11, 231 Neoadjuvant��20, 21, 24–26, 31–33 Nerve growth factor (NGF)��141–147 Network analysis�� 171–187, 342 N-glycan��353, 355, 357–361, 363 N-isopropylacrylamide (NIPAm)��108 Nitrocellulose�� 53, 56, 58, 63, 66, 107, 109, 152, 156–159, 161, 165–167 Nondisclosure agreement (NDA)�� 385, 386 Non-fat dry milk��136 Non-Hodgkin’s lymphoma��28 Notebook��386 Nottingham grade��12 Nuclease-free water��195–197, 208, 221, 225, 228 Nucleotides��40, 91, 195, 220, 225, 227, 228, 230, 391 O Oligosaccharide�� 353, 354, 361 Outer surface protein A (OspA)��135 Outliers�� 183, 341, 344, 349 Ovarian cancer�� 3, 12–15, 383 Overall survival (OS)��20–25 P Packaging�� 404, 406 Paraffin�� 89–92, 94–98, 100, 117, 128, 220, 237, 239–243, 252, 253, 255, 265–279, 283 Paraffin block��89–91, 95, 97, 100, 242 Patent design patents�� 380, 381 first-to-file��384 first-to-invent��384 patent abstract��390 patent application�� 380, 383–392, 400 patent claims�� 379, 383, 388–391 patent drawings��390–391 patent filing�� 381, 391, 392, 395 patent infringement��393–394 patent law��380, 383, 384, 393, 396 patent pending��394 plant patent�� 380, 381 US Patent and Trademark Office (USPTO)��379, 383, 392–394 utility patents��384, 387, 392, 400, 406 Pathological complete response (pCR) RECIST criteria��23 Response Evaluation Criteria in Solid Tumors (RECIST)��23 Pathway analysis��172 Patient population�� 22, 375 Peak Scanner™ software�� 208, 212 Personnel�� 47, 371, 373, 377 Pertuzumab�� 25–26, 33 Phase I��19 Phase II�� 19, 20, 22, 24, 26, 30, 31 Phase III��19, 20, 25, 26, 32 Phenom�� 73–81, 83 Phosphoproteins phosphopeptides��307 Physician-patient relationship informed consent�� 40–42, 45, 47 Jimmy Carter��38, 39 Ulysses Grant��38 Polymer depletion��130 Polymerase��193–196, 200, 208, 211 Polymerase chain reaction (PCR) dPCR��194 melt curve analysis�� 215, 216 PCR master mix�� 196, 197, 200, 227 qPCR�� 215, 216 Polyvinylidene fluoride (PVDF)��56, 58, 59, 61, 63, 64, 66, 67, 137–139 Poorly differentiated��3, 5, 6, 8, 12, 15 Posttranslational modifications�� 55, 313 Pre-analytical variability�� 85–87, 239 Precipitation polymerization��108 Preservative��89, 97 Primer forward primer�� 211, 215 oligonucleotide primers�� 194, 196, 197 primer design�� 194, 195, 199 primer length��195 reverse primer��195 Principal component analysis (PCA)�� 183, 341–343, 346–347, 350 Principal investigator (PI)�� 371, 375 Product development��399–406 Prognosis��7, 13, 14, 24, 47 Progression-free survival (PFS)�� 21, 22, 25 Proprietary information�� 395, 396 Prostate cancer prostatic intraepithelial neoplasia��7 Protease inhibitors�� 118–120, 127, 151 Proteases�� 96, 104, 105, 119, 268, 272, 362 Protein database��309, 316–317, 321–322, 326 Protein denaturation buffer��354 Protein extraction buffer��55, 57, 118, 126, 131, 151 Proteinase K�� 198, 208, 210, 221, 224, 231, 232 Proteomics�� 172, 185, 297, 341 Provisional patent application�� 384, 385 Psammoma bodies��13 Public–private partnerships�� 399, 400 Q Quality��29, 65, 71, 72, 78, 83, 86, 87, 123, 125, 144, 145, 156, 166, 178, 195, 216, 231, 239, 241, 249–252, 255, 267, 271, 277, 283, 285, 287, 291–294, 371, 374–376, 399 Quality of life (QOL)��21, 22 Quantitation�� 313–315, 318, 319, 322, 325, 326, 329, 330, 354, 359, 362, 365 R R Analysis��346–348 Reblot�� 152, 159, 166 Receiver operating characteristics (ROC) curve�� 343, 348–349 Regulatory��20, 21, 46, 52, 180–181, 399, 401, 403–405 Reimbursement�� 44, 402–403 Remazol Brilliant Blue��107–109 Response rate (RR)�� 21–23, 30, 361 Reverse phase (RP) chromatography��356 Reverse phase protein microarray (RPPA) arrayer�� 156, 157, 159 reverse-phase protein array�� 126, 171–187 RPPA data�� 172–181, 185, 186 RIPA buffer��65 RNA��15, 33, 40, 73, 86, 88, 89, 117–119, 125–128, 130, 193, 200, 220, 250 RNase-free�� 98, 195, 196, 200 Running buffer��55, 57, 58, 65, 66, 136, 196, 200, 201 Molecular Profiling: Methods and Protocols 413 Index S Scale-up��403–405 Scanning electron microscopy (SEM) conductive�� 72–74, 79, 82, 83 coolstage�� 72, 74–76, 80, 82 desiccated�� 72–74, 77, 80, 160 electron bombardment��71, 83 sputter coater��72 wet��72 Selumetinib��28 Sensitivity��40, 52, 63, 66, 68, 103, 105, 107, 111, 141, 142, 144, 145, 150, 154, 160, 161, 225, 294, 299, 324, 330, 343, 353, 357 Shrimp alkaline phosphatase (SAP)�� 220, 225, 227–228, 233 Signaling pathway�� 33, 86, 171, 176 Silanized�� 128, 267, 270, 271 Single-nucleotide polymorphisms (SNPs)�� 91, 220, 232 Size-exclusion chromatography�� 362, 365 Somatic mutations�� 219, 220, 231–233 Sorafenib��23 Specificity��40, 51, 52, 54, 64, 164, 194, 314, 321, 343 SpectroCHIP�� 222, 229, 230 Spreadsheet program�� 175, 177, 179 Stable isotope��313–315, 317, 319, 322–324, 329, 353–365 Standard curves��144, 145, 147, 150, 152, 156, 317, 322–324, 328, 329 Statistical�� 30, 41, 163, 172, 179, 182, 341, 343, 354, 375 Sypro Ruby protein blot stain�� 153, 156, 159, 161–163, 167 T Taq DNA polymerase��194 Thermocycler��196, 197, 201, 208, 211 Thionin�� 117–120, 127 Time to progression (TTP)��21, 22 Tissue tissue processing�� 85, 89–97, 236 tissue section cutting�� 221–223, 267, 270–271 Tissue-Tek VIP tissue processor��89 TMB (3,3’,5,5’-tetramethylbenzidine)�� 142, 143, 145, 146 TNM classification system��15 Transfer buffer�� 56, 58, 59, 65, 66, 136–138 Translational research�� 51, 52, 367, 406 Trastuzumab�� 25, 32, 33 Travel�� 232, 287, 293, 373 Tris-acetate-EDTA (TAE)��196 Tris-borate-EDTA (TBE)�� 202, 208, 212 Tris-buffered saline (TBS)�� 56, 57, 152, 161 Tris buffered saline with tween (TBST)�� 152, 161 TRYPHAENA��26 Molecular Profiling: Methods and Protocols 414 Index Trypsin�� 269, 274, 298, 299, 301, 303, 304, 306, 314–316, 318, 320–322, 326, 327 Tumor clinical stage��2 pathologic stage��2 residual cancer burden (RCB)��24–25 staging and grading��1–15 tumor grade��3 tumor stage��2–3 Validation�� 33, 51–69, 86, 173 Visualization��88, 116, 121, 129, 165, 172, 175, 176, 178, 180–181, 260, 342 U X UV transilluminator�� 153, 164 UV-Vis�� 143, 145 Xylene��89–92, 96–99, 118, 120, 129, 196, 221, 223, 224, 237, 238, 242, 245, 255, 267, 271, 272, 277 Xylene substitute��90, 96, 98, 221, 224 V Vacuum��54, 56, 59, 61, 62, 68, 71, 72, 75, 82, 90, 91, 93, 94, 107, 108, 220, 229, 237, 300, 316, 320, 323, 326, 356–358 W Well-differentiated�� 3–6, 8, 15 Western blot SNAP i.d.�� 56–57, 59–62, 68 World Trade Organization�� 381, 382, 384 Z ZipTip�� 301, 305–307, 316, 318–320, 323–326 ... between N0 and N3, and Virginia Espina (ed.), Molecular Profiling: Methods and Protocols, Methods in Molecular Biology, vol 1606, DOI 10.1007/978-1-4939-6990-6_1, © Springer Science+Business Media... studied and therapeutic agents have been evaluated Virginia Espina (ed.), Molecular Profiling: Methods and Protocols, Methods in Molecular Biology, vol 1606, DOI 10.1007/978-1-4939-6990-6_2, © Springer... genomic profiling, technologies which in the intervening years have rapidly evolved into routine clinical assays for detecting mutations Mass spectrometry for protein profiling has also evolved into

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