Table Of ContentGuidelines 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
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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
