Guidelines for Molecular Analysis in Archive Tissues PDF
Preview Guidelines for Molecular Analysis in Archive Tissues
Guidelines for Molecular Analysis in Archive Tissues Giorgio Stanta (Editor) Guidelines for Molecular Analysis in Archive Tissues Editor Prof. Dr. Giorgio Stanta Department of Medical Sciences University of Trieste Cattinara Hospital Strada di Fiume 447 Trieste Italy [email protected] ISBN 978-3-642-17889-4 e-ISBN 978-3-642-17890-0 DOI 10.1007/978-3-642-17890-0 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2011925943 © Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, 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. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and appli- cation contained in this book. In every individual case the user must check such information by consulting the relevant literature. Cover design: eStudioCalamar, Figueres/Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Preface These guidelines are devoted to disseminate molecular methods that can be used to analyze DNA, RNA, and proteins in formalin-fixed and paraffin-embedded (FFPE) tissues. We have called them guidelines because this is the first method book that is entirely dedicated to archive tissues. They are addressed to pathologists, biologists, and biotechnologists who do research in FFPE tissues. The protocols presented in these guidelines derive from the experience of the laboratories participating in the European project “Archive tissues: improving molecular medicine research and clini- cal practice” – IMPACTS (www.impactsnetwork.eu). The 20 participants, from 11 European countries, are some of the most experienced groups in molecular analysis of fixed and paraffin-embedded human tissues. Among them are some of the groups that developed this type of molecular analysis for the first time. These guidelines are mostly dedicated to the homemade protocols that were devel- oped, tested, and even validated by multicentric analyses performed by the IMPACTS groups. Experience in basic molecular methods is necessary to develop molecular pathology activities in human tissues. This allows research without too many techni- cal limitations. In this way the researcher is also capable to better evaluate commer- cial kits and validate them. Also commercial kits are reported in some protocols, especially when the authors have consolidated habits in kit utilization or when advanced techniques are used without being directly developed by the participants. Thus the reported commercial reagents and kits reflect in the same way the experi- ence of the IMPACTS laboratories. However, in our experience standardization of molecular methods by commercial kits is not sufficient to guarantee reproducibility; good laboratory practice is absolutely necessary to obtain reliable results. Sometimes a slightly different “lab jargon” is maintained in the different chapters; this reflects the authors’ consuetude but it does not compromise its clarity. Basic footnotes are repeated in many chapters to make it easy to access the single protocol. We recognize that semi- or fully automated instruments for nucleic acids extrac- tion from FFPE could be very useful in establishing standardization and method reproducibility. In the nearest future they will represent an important task for any molecular pathology laboratory devoted to routine diagnostic molecular analyses in FFPE. However, we did not mention them in these guidelines, because we did not have the chance to compare the performances of the different commercial options. These guidelines are divided into different parts related to tissue processing and macromolecule preservation, molecular analysis of DNA, RNA, and proteomics, and a short chapter about internal quality control. Small chapters about the new develop- ing technologies are also included, these are often confined to the major research centres, but, as past experience has showed us, they sometimes diffuse very quickly. v vi Preface Also a chapter dedicated to forensic methods has been added. Since nucleic acid degradation is a common problem with FFPE tissues, useful suggestions can be obtained also from this issue together with specimen identification through DNA analysis. Most of the methods here reported are more or less similar to those used in fresh cells and tissues, but the modifications made in the protocols are necessary to obtain a positive result in human FFPE tissues. In the intention of the authors all the methods and protocols are described for a laboratory direct use, and the addition of explanatory notes should help even less experienced researchers, but a basic experience of laboratory research methods is required. If during the reading and practical use of the protocols errors or unclear and insufficient explanations are found, please contact directly the IMPACTS Group by e-mail ([email protected]). Bioethical norms are of pivotal importance in the use of human tissues for research, but we avoided ethical considerations because of the lack of uniformity in the direc- tives on clinical residual tissue research in European countries. The only general suggestion is to contact the local ethical committees. I would like to thank very much all the contributors of the IMPACTS group that collaborated not only in the preparation of the chapters but also in the multicentric project of methods validation, and took part in the extensive discussions held within the IMPACTS meetings. I would like to thank Serena Bonin, who has worked with me for the past fifteen years, for her continuous interest and effort in developing molecular methods in archive tissues. I would like to specially mention Isabella Dotti for the work done in the prepara- tion of the DNA and RNA methods, and Valentina Faoro for the groundwork and the assembling of the proteomics chapters. This book could have not been edited without the continuous effort of Valentina Melita, Danae Pracella, and Renzo Barbazza who dedicated a lot of time to reading, correcting, and improving the comprehension and the presentation of the protocols. Trieste, January 2011 Giorgio Stanta Contents Part I Archive Tissues 1 Archive Tissues............................................. 3 Giorgio Stanta 2 Preanalytical Time Interval (PATI) and Fixation................. 5 Lorenzo Daniele, Giuseppe D’Armento, and Gianni Bussolati 3 Formalin-Free Fixatives ..................................... 13 Isabella Dotti, Serena Bonin, Giorgio Basili, and Valentina Faoro Part II Dissection of Tissue Components 4 Manual Microdissection ..................................... 21 Valentina Faoro and Giorgio Stanta 5 Tissue Microarray (TMA).................................... 23 Valentina Faoro and Anna Sapino 6 Laser Capture Microdissection (LCM) ......................... 27 Elvira Stacher, Hannelore Kothmaier, Iris Halbwedl, and Helmut H. Popper Part III E xtraction, Purification, Quantification and Quality Assessment of Nucleic Acids 7 DNA Extraction from Formalin-Fixed Paraffin-Embedded (FFPE) Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Serena Bonin, Patricia J.T.A. Groenen, Iris Halbwedl, and Helmut H. Popper 8 DNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues (FFPE) (from Small Fragments of Tissues or Microdissected Cells)........................................ 37 Serena Bonin, Patricia J.T.A. Groenen, Iris Halbwedl, and Helmut H. Popper vii viii Contents 9 Fast Protocol for DNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues .................................. 41 Falk Hlubek and Andreas Jung 10 DNA Extraction from Blood and Forensic Samples............... 45 Solange Sorçaburu Cigliero, Elisabetta Edalucci, and Paolo Fattorini 11 Restoration and Reconstruction of DNA Length ................. 55 Serena Bonin and Federica Tavano 12 RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues .................................. 57 Serena Bonin and Giorgio Stanta 13 RNA Extraction from Decalcified and Non-decalcified Formalin-Fixed Paraffin-Embedded Tissues..................... 63 Marco Alberghini, Stefania Benini, Gabriella Gamberi, Stefania Cocchi, and Licciana Zanella 14 RNA Temperature Demodification............................. 67 Serena Bonin and Giorgio Stanta 15 MicroRNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues .................................. 71 Roberto Cirombella and Andrea Vecchione 16 Quantification of Nucleic Acids ............................... 75 Isabella Dotti and Serena Bonin 17 Integrity Assessment of Nucleic Acids.......................... 81 Isabella Dotti and Serena Bonin 18 DNase Treatment of RNA .................................... 87 Isabella Dotti and Serena Bonin Part IV Reverse Transcription (RT) 19 Reverse Transcription ....................................... 93 Isabella Dotti, Serena Bonin, and Ermanno Nardon Part V Nucleic Acid Amplification 20 General Protocol for End-Point PCR........................... 101 Serena Bonin and Isabella Dotti 21 Quantitative End-Point PCR ................................. 105 Serena Bonin and Isabella Dotti Contents ix 22 Nested-PCR ............................................... 111 Serena Bonin and Isabella Dotti 23 General Protocol for Dot-Blot................................. 115 Serena Bonin and Isabella Dotti 24 PCR-ELISA ............................................... 117 Christiane Schewe, Wilko Weichert, and Manfred Dietel 25 Quantitative Real-Time RT-PCR .............................. 121 Isabella Dotti, Ermanno Nardon, Danae Pracella, and Serena Bonin Part VI DNA Sequencing 26 DNA Sequencing from PCR Products .......................... 135 Giorgio Basili, Serena Bonin, and Isabella Dotti 27 Pyrosequencing Analysis for Detection of KRAS Mutation ........ 143 Gerlinde Winter and Gerald Höfler Part VII Microsatellite Analysis 28 Microsatellite Instability (MSI) Detection in DNA from FFPE Tissues .............................................. 155 Damjan Glavacˇ and Ermanno Nardon 29 General Protocol for Loss of Heterozygosity Detection ............ 171 Damjan Glavacˇ and Ermanno Nardon Part VIII Methylation Analysis 30 Qualitative Methylation Status Assessment ..................... 181 Damjan Glavacˇ and Ermanno Nardon 31 Quantitative Methylation Status Assessment in DNA from FFPE Tissues with Bisulfite Modification and Real-Time Quantitative MSP........................................... 193 Ermanno Nardon Part IX Analysis of Copy Number Variations 32 Comparative Genomic Hybridisation (CGH).................... 203 Hannelore Kothmaier, Elvira Stacher, Iris Halbwedl, and Helmut H. Popper 33 Multiplex Ligation-dependent Probe Amplification (MLPA) ....... 215 Ana Pilar Berbegall, Eva Villamón, Samuel Navarro, and Rosa Noguera
The list of books you might like
