M e t h o d s i n Mo l e c u l a r Bi o l o g y

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1 M e t h o d s i n Mo l e c u l a r Bi o l o g y Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes:

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3 Gene Expression Profiling Methods and Protocols Second Edition Edited by Lorraine O Driscoll School of Pharmacy and Pharmaceutical Sciences,

4 Editor Lorraine O Driscoll School of Pharmacy and Pharmaceutical Sciences Trinity College Dublin Dublin, Ireland lodrisc@tcd.ie ISSN e-issn ISBN e-isbn DOI / Springer New York Dordrecht Heidelberg London Library of Congress Control Number: Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (

5 Preface It was with great pleasure that I accepted the invitation to be Editor for this volume of Methods in Molecular Biology. This book collates chapters by experts on a wide range of topics relevant to gene expression profiling. Understanding gene expression and how it changes under normal and pathological conditions is essential to our understanding of the fundamentals of cell biology through to the targeted treatment of disease. This book compiles protocols, written by experts in their respective fields, for a broad range of techniques, currently available and being further developed, for the analysis of gene expression at the DNA, RNA, and protein levels. In summary, the topics addressed in this volume of Methods in Molecular Biology span the field of gene expression from basic to advanced methodologies, including step-by-step protocols which the reader can easily follow, as well as trouble-shooting tips and tricks to help ensure the success of their application. The chapter by Gurvich and Skoblov brings us through the fundamentals of polymerase chain reaction (PCR), extending to multiplex approaches. Rani and O Driscoll advance on this strong basis to the application of PCR for the analysis of specimens where very limited amounts of starting material exist, i.e., in the extracellular environment. Considering the importance of being able to globally co-analyze all mrnas transcribed, Mehta outlines microarray methods. Following microarray analysis, researchers often find progression to the stage of being able to correctly analyze their data to be a substantial challenge. For this reason, Mehta and Rani proceed to explain software and tools for the analysis of such datasets. Friel, Crown, and O Driscoll detail gene expression analysis specifically in relation to cancer cells and circulating tumor cells. As formalinfixed paraffin-embedded tissue is quite extensively available for gene expression studies, relative to the availability of fresh/frozen tissue, April and Fan have described methods for such studies, using the whole genome DASL assay. Moving into the world of micrornas (mirnas), Hennessy and O Driscoll outline methods for their determination, including procedures which are applicable to the assessment of both intracellular and extracellular mirnas. The final steps in our journey through profiling of gene expression involve analysis at the protein level. Here, initially considering extracted proteins, Germano and O Driscoll describe basic, but essential, methods for assessing proteins by Western blotting, while Meleady advances this to more global protein studies that involve 2D gel electrophoresis and mass spectrometry technologies. A powerful advancement for in situ protein evaluation is our ability to construct user-friendly arrays representative of cell lines and/or tissues of interest. Experts in this field, Gately, Kerr, and O Byrne, detail their optimized methods for design, construction, and analysis of such cell/tissue arrays. Basic and advanced immunohistochemistry and immunofluorescence techniques are clearly described by Katikreddy and O Sullivan, while Hanrahan, Harris, and Egan detail laser scanning confocal microscopy in such a way as to make it less daunting to consider, when analysis and co-analysis of proteins in situ is desired. Rani, O Brien, Kelleher, Corcoran, Germano, Radomski, Crown, and O Driscoll describe procedures for the isolation of exosomes and their subsequent gene expression analysis as potential representative of the cells from which they have been secreted. v

6 vi Preface Finally, Buckley, Davies, and Ehrhardt take us through step-by-step approaches to the successful application of atomic force microscopy and high-content analysis for analyzing expression of particular genes of interest. I hope that this collection of clearly described and illustrated chapters will be helpful to researchers in academia, in hospitals, and in industry who are interested in applying techniques, whether they be very basic or very advanced, for the analysis of gene expression. I also hope this will be of use to those who want to review progress in this very exciting, evolving field. Mostly I hope it will contribute, in some way, to a better understanding of gene expression changes that occur under normal and pathological conditions which can advance progress toward better treatments for those suffering from disease. Dublin, Ireland Lorraine O Driscoll

7 Contents Preface.... Contributors.... v ix 1 Real-Time PCR and Multiplex Approaches... 1 Olga L. Gurvich and Mikhail Skoblov 2 Reverse-Transcriptase Polymerase Chain Reaction to Detect Extracellular mrnas Sweta Rani and Lorraine O Driscoll 3 Microarray Analysis of mrnas: Experimental Design and Data Analysis Fundamentals Jai Prakash Mehta 4 Software and Tools for Microarray Data Analysis Jai Prakash Mehta and Sweta Rani 5 Analysis of Gene Expression as Relevant to Cancer Cells and Circulating Tumour Cells Anne M. Friel, John Crown, and Lorraine O Driscoll 6 Gene Expression Profiling in Formalin-Fixed, Paraffin-Embedded Tissues Using the Whole-Genome DASL Assay Craig S. April and Jian-Bing Fan 7 MicroRNA Expression Analysis: Techniques Suitable for Studies of Intercellular and Extracellular MicroRNAs Erica Hennessy and Lorraine O Driscoll 8 Western Blotting Analysis as a Tool to Study Receptor Tyrosine Kinases Serena Germano and Lorraine O Driscoll 9 2D Gel Electrophoresis and Mass Spectrometry Identification and Analysis of Proteins Paula Meleady 10 Design, Construction, and Analysis of Cell Line Arrays and Tissue Microarrays for Gene Expression Analysis Kathy Gately, Keith Kerr, and Ken O Byrne 11 Immunohistochemical and Immunofluorescence Procedures for Protein Analysis Kishore Reddy Katikireddy and Finbarr O Sullivan 12 Advanced Microscopy: Laser Scanning Confocal Microscopy Orla Hanrahan, James Harris, and Chris Egan 13 Isolation of Exosomes for Subsequent mrna, MicroRNA, and Protein Profiling Sweta Rani, Keith O Brien, Fergal C. Kelleher, Claire Corcoran, Serena Germano, Marek W. Radomski, John Crown, and Lorraine O Driscoll vii

8 viii Contents 14 Atomic Force Microscopy and High-Content Analysis: Two Innovative Technologies for Dissecting the Relationship Between Epithelial Mesenchymal Transition-Related Morphological and Structural Alterations and Cell Mechanical Properties Stephen T. Buckley, Anthony M. Davies, and Carsten Ehrhardt Index

9 Contributors Cr a i g S. Ap r i l Illumina, Inc., San Diego, CA, USA St e p h e n T. Bu c k l e y School of Pharmacy & Pharmaceutical Sciences, Cl a i r e Co r c o r a n School of Pharmacy & Pharmaceutical Sciences & MTCI, Jo h n Cr o w n Molecular Therapeutics for Cancer Ireland (MTCI), Dublin City University, Dublin, Ireland An t h o n y M. Dav i e s Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland Ch r i s Eg a n Smurfit Institute of Genetics, Ca r s t e n Eh r h a r d t School of Pharmacy & Pharmaceutical Sciences, Ji a n-bi n g Fa n Illumina, Inc., San Diego, CA, USA An n e M. Fr i e l School of Pharmacy and Pharmaceutical Sciences & MTCI, Ka t h y Gat e l y Department of Clinical Medicine, Thoracic Oncology Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, Dublin, Ireland Se r e n a Ge r m a n o School of Pharmacy and Pharmaceutical Sciences, Ol g a L. Gu r v i c h Moscow State University, Moscow, Russia Or l a Ha n r a h a n School of Biochemistry & Immunology, Trinity College Dublin, Dublin, Ireland Ja m e s Ha r r i s School of Biochemistry & Immunology, Trinity College Dublin, Dublin, Ireland Er i c a He n n e s s y National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland Ki s h o r e Re d d y Ka t i k i r e d d y National Institute of Cellular Biotechnology, Dublin City University, Dublin, Ireland Ke i t h Ke r r Department of Pathology, Aberdeen University Medical School & Aberdeen Royal Infirmary, Foresterhill, Aberdeen, UK Fe r g a l C. Ke l l e h e r Department of Medical Oncology, St. Vincent s University Hospital, Dublin, Aberdeen, UK Ja i Pr a k a s h Me h t a Conway Institute, University College Dublin, Dublin, Ireland Pa u l a Me l e a d y National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland Ke i t h O Br i e n School of Pharmacy & Pharmaceutical Sciences, Ke n O By r n e Department of Clinical Medicine, Thoracic Oncology Research Group, Institute of Molecular Medicine, Trinity Centre for Health Sciences, Dublin, Ireland ix

10 x Contributors Lo r r a i n e O Dr i s c o l l School of Pharmacy and Pharmaceutical Sciences, Fi n b a r r O Su l l i va n National Institute of Cellular Biotechnology, Dublin City University, Dublin, Ireland Ma r e k W. Ra d o m s k i School of Pharmacy & Pharmaceutical Sciences, Sw e t a Ra n i School of Pharmacy & Pharmaceutical Sciences, Panoz Institute, Mikhail Sk o b l o v Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia