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Cardiac Gene Expression
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M E T H O D S I N M O L E C U L A R B I O L O G Y™

Cardiac Gene
Expression
Methods and Protocols

Edited by

Jun Zhang
Division of Cardiology, CVRL/Geffen School of Medicine at UCLA,
Los Angeles, CA
and

Gregg Rokosh
Division of Cardiology, University of Louisville,
Louisville, KY
© 2007 Humana Press Inc.
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Cover illustration: From Fig. 5D (Background) and Fig. 6D (Inset) of Chapter 9, “In Situ Hybridization: A
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Cardiac gene expression : methods and protocols / edited by Jun Zhang and Gregg Rokosh.
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1. Myocardium—Laboratory manuals. 2. Gene expression—Laboratory manuals. 3. Genetic
regulation—Laboratory manuals. I. Zhang, Jun, 1968- II. Rokosh, Gregg. III. Series: Methods
in molecular biology (Clifton, N.J.) ; v. 366.
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Preface
The past decade has ushered in enormous changes in how we perceive and
study changes in gene expression in the heart. Early in the 1990s, the human
genome project was just getting underway and establishing methods with the
sensitivity to measure changes in the expression of genes with low copy number
was an accomplishment. We all experienced some trepidation when the first
news of microarrays arrived espousing the ability to measure changes in expres-
sion of hundreds to tens of thousands of genes (the whole genome) at once. This
high throughput method was an astonishing jump in our approach to biological
science. At the same time Steve Fodor and Pat Brown published papers describ-
ing two completely different approaches to measuring the expression changes
of large numbers of genes at the same time. Thus began the microarray era and
as a consequence the beginning of an era with a host of new approaches in
pursuit of understanding the role and regulation of gene expression in cell biol-
ogy and pathology including driving forward the field of bioinformatics.
The array, no pun intended, of contributions contained in Cardiac Gene
Expression: Methods and Protocols an edition of Humana’s Methods in Molecu-
lar series, address both new and established methods that researchers in the car-
diac field will certainly find useful as a reference for the development of projects
and training. Our aim in this compilation was to provide insight and details for
a comprehensive range of methods that will serve both startup and sophisticated
users alike. Sections cover expression profiling by microarray Section I, tar-
geted analysis of gene expression (Section II), transcription factor DNA bind-
ing and regulation of promoter activity (Section III), in silico approaches to
identifying functional cis regulatory elements and regulation of cardiac gene
expression (Section IV), in silico and mass spectrometry methods to identify
sequence nucleotide polymorphisms (SNPs) (Section V), and to bring findings
from the above studies to the next level overexpression of genes in vivo and
isolated myocytes and cardiac-specific targeted gene deletion (Section VI).
Section I, Cardiac Gene Expression Profiling: the Global Perspective. Five
chapters describe several different approaches to examining and identifying
changes in gene expression in the transcriptome as well as analytic approaches.
Methods and analysis have improved significantly as many investigators have
strived to increase array reliability and reproducibility. Section II, Cardiac Gene
Regulation: Gene-Specific mRNA Measurement in the Myocardium, follows
accordingly with chapters outlining more sensitive and gene targeted expres-
sion methods that are more conducive for follow up studies to verify and fur-

v
vi Preface

ther characterize those important findings from array experiments or those of

your favorite gene. Underlying mechanisms of gene regulation can be studied
using methods that focus on the interaction of transcription factors with their
cognate cis binding elements and how these cis elements impact overall pro-
moter activity in Section III, Cardiac Gene Regulation: Promoter Character-
ization in the Myocardium. Changes in gene expression reflect the combined
effects of transcriptional enhancers and repressors that serve to precisely con-
trol the level of expression of thousands of genes from conception to death.
Studies that focus on how the interaction between transcription factors and
their cognate cis DNA elements regulate gene expression were provided some
assistance recently with the completion of the Human Genome Project in 2003
in addition to several other genomes with more coming available at a rapid
pace. New analytical approaches to decipher the functional elements buried in
the 3 billion nucleotides of the human genome and other model genomes are
described in Sections IV, In Silico Assessment of Regulatory cis-Elements
and Gene Regulation, and V, Cardiac Single Nucleotide Polymorphisms. One
important aspect of understanding the importance of available sequence is
being able to sift through sequence and reliably identify and distinguish func-
tional regulatory elements from nonfunctional elements. Pennachio and col-
leagues at Lawrence Livermore Labs have simplified this task by using a
comparative approach. By using available genome sequence for several differ-
ent species across evolution this approach was able to reliably predict the func-
tionality of elements according to their evolutionary conservation. Resources
for the analysis of gene regulation data and SNPs will provide essential func-
tionality for the understanding of changes in gene expression and effects of
SNPs on gene function and expression. With the identification of exciting new
targets one begins to think of the functional aspects and begins to plan experi-
ments to validate hypotheses. Section VI, Gene Overexpression and Targeting
in the Myocardium, highlights methods that facilitate overexpression or car-
diac specific targeted deletion of your favorite gene in the heart
Thus, this array of contributions provides an array of methods that will take
the investigator through screening, analysis, characterization, and functional
confirmation of novel genes or old genes with a new function serving as a
template for a solid research program.
Jun Zhang
Gregg Rokosh
Contents
Preface .............................................................................................................. v
Contributors ..................................................................................................... ix

PART I. CARDIAC GENE EXPRESSION:

THE GLOBAL PERSPECTIVE
1 Microarray Analysis of Gene Expression in Murine
Cardiac Graft Infiltrating Cells
Yurong Liang, Xin Lu, and David L. Perkins ......................................... 3
2 Expression Profiling Using Affymetrix GeneChip®
Probe Arrays
Martina Schinke-Braun and Jennifer A. Couget ................................. 13
3 Serial Analysis of Gene Expression (SAGE):
A Useful Tool to Analyze the Cardiac Transcriptome˚˚˚˚
Kirill V. Tarasov, Sheryl A. Brugh, Yelena S. Tarasova,
and Kenneth R. Boheler .................................................................. 41
4 Functional Genomics by cDNA Subtractive Hybridization
Christophe Depre ................................................................................ 61
5 Statistical Methods in Cardiac Gene Expression Profiling:
From Image to Function
Sek Won Kong .................................................................................... 75

PART II. CARDIAC GENE REGULATION:

GENE-SPECIFIC MRNA MEASUREMENT IN THE MYOCARDIUM
6 Measurement of Cardiac Gene Expression by Reverse
Transcription Polymerase Chain Reaction (RT-PCR)
Nicola King ....................................................................................... 109
7 Quantitative (Real-Time) RT-PCR in Cardiovascular Research
Kevin John Ashton and John Patrick Headrick ................................. 121
8 RNase Protection Assay for Quantifying Gene
Expression Levels
Yongxia Qu and Mohamed Boutjdir ................................................. 145
9 In Situ Hybridization: A Technique to Study Localization
of Cardiac Gene Expression
Thierry P. Calmels and David Mazurais ........................................... 159

vii
viii Contents

PART III. CARDIAC GENE REGULATION:

PROMOTER CHARACTERIZATION IN THE MYOCARDIUM
10 Characterization of cis-Regulatory Elements and Transcription
Factor Binding: Gel Mobility Shift Assay
Jim Jung-Ching Lin, Shaun E. Grosskurth, Shannon M. Harlan,
Elisabeth A. Gustafson-Wagner, and Qin Wang .......................... 183
11 Mapping Transcriptional Start Sites and In Silico DNA
Footprinting
Martin E. Cullen and Paul J. R. Barton ............................................. 203
12 Characterization of Cardiac Gene Promoter Activity:
Reporter Constructs and Heterologous Promoter Studies
Hsiao-Huei Chen and Alexandre F. R. Stewart ................................ 217

PART IV. IN SILICO ASSESSMENT OF REGULATORY CIS-ELEMENTS

AND GENE REGULATION
13 Comparative Genomics:
A Tool to Functionally Annotate Human DNA
Jan-Fang Cheng, James R. Priest, and Len A. Pennacchio ................ 229
14 Developing Computational Resources in Cardiac Gene
Expression
Michael B. Bober and Raimond Winslow ......................................... 253

PART V. CARDIAC SINGLE NUCLEOTIDE POLYMORPHISMS

15 In Silico Analysis of SNPs and Other High-Throughput Data
Neema Jamshidi, Thuy D. Vo, and Bernhard O. Palsson ................. 267
16 Discovery and Identification of Sequence Polymorphisms
and Mutations with MALDI-TOF MS
Dirk van den Boom and Mathias Ehrich ........................................... 287

PART VI. GENE OVEREXPRESSION AND TARGETING IN THE MYOCARDIUM

17 Conditional Targeting: Inducible Deletion by Cre Recombinase
Kelly R. O’Neal and Ramtin Agah .................................................... 309
18 Cardiomyocyte Preparation, Culture, and Gene Transfer
Alexander H. Maass and Massimo Buvoli ......................................... 321
19 Adeno-Associated Viral Vector–Delivered Hypoxia-Inducible
Gene Expression in Ischemic Hearts
Hua Su and Yuet Wai Kan ................................................................ 331
20 Lentivirus-Mediated Gene Expression
Jing Zhao and Andrew M. L. Lever ................................................... 343
Index ............................................................................................................ 357
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Contributors
RAMTIN AGAH • The Program in Human Molecular Biology and Genetics,
University of Utah, Salt Lake City, UT, USA (Present address: Altos
Cardiovascular Institute, Mountain View, CA, USA)
KEVIN JOHN ASHTON • Heart Foundation Research Centre, Griffith University,
Southport, Australia
PAUL J. R. BARTON • Heart Science Centre, National Heart and Lung Institute,
Imperial College London, Harefield, Middlesex, UK
MICHAEL B. BOBER • Division of Medical Genetics, A.I. DuPont Hospital
for Children, Wilmington, DE, USA
KENNETH R. BOHELER • The Laboratory of Cardiovascular Science,
The National Institute on Aging, NIH, Baltimore, MD, USA
MOHAMED BOUTJDIR • VA New York Harbor Healthcare System, SUNY
Downstate Medical Center, Brooklyn, NY, and NYU School of Medicine,
New York, NY, USA
SHERYL A. BRUGH • The Laboratory of Cardiovascular Science, The National
Institute on Aging, NIH, Baltimore, MD, USA
MASSIMO BUVOLI • Department of Molecular Cellular and Developmental
Biology, University of Colorado at Boulder, Boulder, CO, USA
THIERRY P. CALMELS • Bioprojet Biotech, Pharmacology Department, Saint
Grégoire Cedex, France
HSIAO-HUEI CHEN • University of Ottawa Health Research Institute, Ottawa,
Canada
JAN-FANG CHENG • Genomics Division, Lawrence Berkeley National Laboratory,
Berkeley, CA, USA, and Joint Genome Institute, U.S. Department of Energy,
Walnut Creek, CA, USA
JENNIFER A. COUGET • Harvard University Bauer Center for Genomics
Research, Cambridge, MA, USA
MARTIN E. CULLEN • Heart Science Centre, National Heart and Lung Institute,
Imperial College London, Harefield, Middlesex, UK
CHRISTOPHE DEPRE • Cardiovascular Research Institute, University of Medicine
and Dentistry New Jersey, New Jersey Medical School, Newark, NJ, USA
MATHIAS EHRICH • SEQUENOM Inc., San Diego, CA, USA
SHAUN E. GROSSKURTH • Department of Biological Sciences, University
of Iowa, Iowa City, IA, USA
ELISABETH A. GUSTAFSON-WAGNER • Department of Biological Sciences,
University of Iowa, Iowa City, IA, USA

ix
xii Contributors

SHANNON M. HARLAN • Department of Biological Sciences, University of Iowa,

Iowa City, IA, USA
JOHN PATRICK HEADRICK • Heart Foundation Research Centre, Griffith
University, Southport, Australia
NEEMA JAMSHIDI • Department of Bioengineering, University of California,
San Diego, San Diego, CA, USA
YUET WAI KAN • Cardiovascular Research Institute/Department of Medicine/
Department of Laboratory Medicine, University of California, San Francisco,
San Francisco, CA, USA
NICOLA KING • Bristol Heart Institute, Department Clinical Science Medicine
at South Bristol, Faculty of Medicine and Dentistry, University of Bristol,
Bristol, UK
SEK WON KONG • Department of Cardiology, Children’s Hospital Boston,
Harvard Medical School, Boston, MA, USA
ANDREW M. L. LEVER • University of Cambridge, Department of Medicine,
Addenbrooke’s Hospital, Hill Road, Cambridge, UK
YURONG LIANG • Laboratory of Molecular Immunology, Brigham &
Women’s Hospital and Harvard School of Public Health, Harvard
Medical School, MA, USA
JIM JUNG-CHING LIN • Department of Biological Sciences, University of Iowa,
Iowa City, IA, USA
XIN LU • Laboratory of Molecular Immunology, Brigham & Women’s
Hospital and Harvard School of Public Health, Harvard Medical School,
MA, USA
ALEXANDER H. MAASS • Department of Medicine, University of Wuerzburg,
Wuerzburg, Germany
DAVID MAZURAIS • Université de Rennes I, Unité INRA-SCRIBE, Campus
Beaulieu, Rennes Cedex, France
KELLY R. O’NEAL • The Program in Human Molecular Biology and Genetics,
University of Utah, Salt Lake City, UT, USA
BERNHARD O. PALSSON • Department of Bioengineering, University of
California, San Diego, San Diego, CA, USA
LEN A. PENNACCHIO • Genomics Division, Lawrence Berkeley National
Laboratory, Berkeley, CA, USA and Joint Genome Institute, U.S. Department
of Energy, Walnut Creek, CA, USA
DAVID L. PERKINS • Laboratory of Molecular Immunology, Brigham &
Women’s Hospital and Harvard School of Public Health, Harvard
Medical School, MA, USA
JAMES R. PRIEST • Genomics Division, Lawrence Berkeley National Laboratory,
Berkeley, CA, USA and Joint Genome Institute, U.S. Department of Energy,
Walnut Creek, CA, USA
Contributors xiii

YONGXIA QU • VA New York Harbor healthcare System, SUNY Downstate

Medical Center, Brooklyn, NY, USA
MARTINA SCHINKE-BRAUN • Novartis Institutes for BioMedical Research,
Cambridge, MA, USA
ALEXANDRE F. R. STEWART • University of Ottawa Heart Institute, Ottawa,
Canada
HUA SU • Cardiovascular Research Institute/Department of Medicine,
University of California, San Francisco, San Francisco, CA, USA
KIRILL V. TARASOV • The Laboratory of Cardiovascular Science, The National
Institute on Aging, NIH, Baltimore, MD, USA
YELENA S. TARASOVA • The Laboratory of Cardiovascular Science,
The National Institute on Aging, NIH, Baltimore, MD, USA
DIRK VAN DEN BOOM • SEQUENOM Inc., San Diego, CA, USA
THUY D. VO • Department of Bioengineering, University of California,
San Diego, San Diego, CA, USA
QIN WANG • Department of Pharmacology, Vanderbilt University Medical
Center, Nashville, TN, USA
RAIMOND WINSLOW • Center for Cardiovascular Bioinformatics and Modeling,
Johns Hopkins University, and The Whitaker Biomedical Engineering
Institute, Baltimore, MD, USA
JING ZHAO • Department of Medicine, Addenbrooke’s Hospital, University
of Cambridge, Hill Road, Cambridge, UK
 Microarray Analysis of Gene Expression 1

CARDIAC GENE EXPRESSION

THE GLOBAL PERSPECTIVE

01/Perkins/1-12 1 12/20/06, 12:12 PM

 2 Liang, Lu, and Perkins

01/Perkins/1-12 2 12/20/06, 12:12 PM

 Microarray Analysis of Gene Expression 3

Microarray Analysis of Gene Expression

in Murine Cardiac Graft Infiltrating Cells

Yurong Liang, Xin Lu, and David L. Perkins

Summary
Microarray technology can rapidly generate large databases of gene expression pro-
files. Our laboratory has applied these techniques to analyze differential gene expression
in cardiac tissue and cells based on mouse heart transplantation. We have analyzed the
different gene expression profiles such as stress or injury including ischemia following
transplantation. We also have investigated the role of infiltrating inflammatory cells dur-
ing graft rejection by purifying subsets of infiltrating cells using GFP transgenic mice
and detailed all technical experiences and issues. The purpose of this study is to assist
researchers to simplify the process of analyzing large database using microarray
technology.
Key Words: Gene expression; microarray, bioinformatics; heart transplantation;
mouse.

1. Introduction
The analysis of gene expression profiles using microarray technology is a
powerful approach to investigate the functions of specific tissues or cells.
Our laboratory has applied these techniques to analyze differential gene
expression in cardiac tissue and cells in a model of murine heart transplanta-
tion (1–4). Specifically, we have analyzed the response by cardiac cells to vari-
ous forms of stress or injury including ischemia following transplantation (5).
In addition, we have investigated the role of infiltrating inflammatory cells
during graft rejection by purifying subsets of infiltrating cells. Using current
microarray technology, it is possible to analyze approx 45,000 probe sets rep-
resenting known mouse genes or expressed sequence tags (ESTs). The ability
to perform global analyses of gene expression creates the potential to analyze

From: Methods in Molecular Biology, vol. 366: Cardiac Gene Expression: Methods and Protocols
Edited by: J. Zhang and G. Rokosh © Humana Press Inc., Totowa, NJ

01/Perkins/1-12 3 12/20/06, 12:12 PM

 4 Liang, Lu, and Perkins

complex biological systems. These methods could be applied to other ques-

tions of cardiac development or disease.

2. Materials
1.
Collagenase II (Gibco) and pancreatin (Sigma).
2.
D-phosphate-buffered saline (PBS; Gibco).
3.
Tri Reagent (Gibco-BRL Life Technologies, Rockville, MD).
4.
Dnase I (Invitrogen).
5.
SuperScript II (Invitrogen).
6.
ALTRA flow cytometer (Beckman Coulter).
7.
SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA).
8.
GeneAmp 5700 Sequence Detection System (Applied Biosystems).
9.
SuperScript Choice system (Gibco-BRL Life Technologies) and T7-(dT) poly-
merase (Gensetoligos, La Jolla, CA).
10. BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farming-
dale, NY).
11. RNeasy mini kit (Qiagen, Valencia, CA).
12. Affymetrix GeneChip Software.

3. Methods
3.1. Vascularized Heterotopic Cardiac Transplantation
1. Murine hearts are transplanted as previously described (6,7).
2. Briefly, hearts are harvested from freshly sacrificed donors and immediately
transplanted into 8- to 12-wk-old recipients that are anesthesized via intraperito-
neal injection with 100 mg/kg of ketamine and 20 mg/kg of xylazine.
3. The donor aorta is attached to the recipient abdominal aorta by end-to-side
anastamosis, and the donor pulmonary artery is attached to the recipient vena
cava by end-to-side anastomosis.
4. All surgical procedures should be completed in less than 45 min from the time
that the donor heart is harvested to ensure similar ischemia times. Donor hearts
that do not beat immediately after reperfusion or that stop within 1 d follow-
ing transplantation should be excluded (>98% of all grafts function at 1 d follow-
ing transplantation).

3.2. Single Cell Suspension

1. Donor grafts are harvested at the indicated time following transplantation and
processed to prepare a single-cell suspension using collagenase and pancreatin
digestion.
2. The graft heart is harvested following cold saline perfusion.
3. Hearts are minced to fine fragments with a scalpel or razor blade.
4. The heart tissue is digested four times with 0.5% collagenase II (Gibco) and
2.5% pancreatin (Sigma) in 37°C water for 7 min (see Note 1).

01/Perkins/1-12 4 12/20/06, 12:12 PM

 Microarray Analysis of Gene Expression 5

5. The cell suspension should be filtered and washed twice with 2% FCS D-PBS
solution.
6. Resuspend cells, add 2 mL 2% FAS solution, and perform flow cytometry analysis.
3.3. Cell Sorting
Graft infiltrating cells have been shown to play important roles in triggering
immune responses during graft rejection after transplantation and other inflam-
matory diseases such as myocarditis. To determine whether gene expression
differences were expressed in infiltrating inflammatory or stromal cells, we
used microarray technology to analyze the gene expression profile. To purify
cell populations of infiltrating or stromal cells, we purified cell subsets by fluo-
rescence-activated cell sorting (FACS) based on expression of green fluores-
cent protein (GFP) or fluorescent labeled monoclonal antibodies (see Note 2).
Gene expression can be analyzed by DNA microarrays or real-time polymerase
chain reaction (PCR) in the purified cell populations.
3.3.1. Analysis of Graft Infiltrating Cells
Because of technical difficulties, methods of purifying infiltrating cells
often isolate a small percentage of the total population of infiltrating cells.
To improve specificity and yield, we have developed a protocol using donor or
recipient mice containing a transgene that constitutively expresses the GFP in
all cells. These cells have greater than three logs of green fluorescence, making
purification by FACS efficient and quantitative. As previously reported, we
can purify sufficient infiltrating cells to perform microarray analysis from small
numbers of mice. For example, our typical yield is from 106 (at early time
points) to 107 (at late time points) infiltrating cells per graft (see Note 3). Thus,
we can harvest sufficient cells from a single mouse at d 7 following transplan-
tation to obtain sufficient RNA for microarray analysis. An advantage of this
approach is that infiltrating cells can be analyzed without requiring amplifica-
tion of RNA.
3.4. RNA Extraction
Total RNA is isolated from tissues or purified cell populations using
TRIZOL reagent (Gibco-BRL Life Technologies). RNA purity is determined
initially by 260/280 = 1.85 to 2.01 and by scanning with an Agilent 2100
Bioanalyzer using the RNA 6000 Nano LabChip®. RNA samples not meeting
these basic parameters of quality should be excluded from the study.
3.5. DNA Microarrays
1. The initial step of cDNA synthesis is performed using Affymetrix protocols with
the T7 dT Primer (100 pM) 5'-GGCCAGTGAATTGTAATACGACTCACTATA
GGGAGGCGG-(dT) 24-3'.

01/Perkins/1-12 5 12/20/06, 12:12 PM

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