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 METHODS IN MOLECULAR BIOLOGY™ • 366 ZHANG
METHODS IN MOLECULAR BIOLOGY™ 366
ROKOSH
SERIES EDITOR: John M. Walker

Cardiac Gene Expression

Cardiac

Cardiac Gene Expression: Methods and Protocols

Methods and Protocols

Gene Expression
Edited by

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

Gregg Rokosh
Division of Cardiology,
Methods and Protocols
University of Louisville, Louisville, KY

Cardiac Gene Expression: Methods and Protocols presents both cutting-edge and established
methods for studying cardiac gene expression. The protocols provide a template for solid research,
Edited by
and cover the process through screening, analysis, characterization, and functional confirmation of
novel genes or known genes with a new function.
Section I, Cardiac Gene Expression Profiling: The Global Perspective, discusses several dif-
ferent approaches to examining, identifying, and analyzing changes in transcriptome gene expres-
Jun Zhang
Gregg Rokosh
sion. Section II, Cardiac Gene Regulation: Gene-Specific mRNA Measurement in the Myocardium,
outlines more sensitive and gene-targeted expression methods. Section III, Cardiac Gene Regulation:
Promoter Characterization in the Myocardium, provides protocols for the study of underlying gene
regulation mechanisms by focusing on the interaction of transcription factors with their cognate cis
binding elements. Section IV, In Silico Assessment of Regulatory cis-Elements and Gene Regulation,
and Section V, Cardiac Single Network Polymorphisms, emphasize new analytical approaches for
deciphering the functional elements buried in the 3 billion nucleotides of the human genome and
other model genomes. The concluding section, Gene Overexpression and Targeting in the Myocar-
dium, highlights methods that facilitate overexpression or cardiac-specific targeted gene deletion.

FEATURES

• Resources for the analysis of gene regulation data and SNPs

• Comparative genomic approach for functionally annotating human DNA
• Quantitative, real-time RT-PCR in cardiovascular research
• In silico analysis of SNPs and other high-throughput data
• Conditional targeting in the myocardium MiMB
366
Methods in Molecular Biology™ • 366
Cardiac Gene Expression: Methods and Protocols
ISBN: 1-58859-352-1 E-ISBN: 1-59745-030-8
ISBN 13: 978-1-58829-352-7 E-ISBN: 978-1-59745-030-0
ISSN: 1064-3745
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Cardiac Gene Expression
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John M. Walker, SERIES EDITOR

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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
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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
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612.1’7—dc22 2006008866
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
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

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 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'.

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 6 Liang, Lu, and Perkins

2. In vitro transcription and preparation of labeled RNA is performed using the Enzo
BioArray High Yield RNA Transcription Labeling Kit.
3. The in vitro transcription sample is cleaned with standard Affymetrix protocols
and quantified on a Bio-Tek UV Plate Reader.
4. Twenty micrograms of in vitro transcription material is the nominal amount
hybridized to the GeneChip® arrays, an amount easily obtainable from graft
tissue.
5. The arrays are incubated in a model 320 (Affymetrix) hybridization oven at a
constant temperature of 45°C overnight.
6. Preparation of the microarray for scanning is performed with Affymetrix wash
protocols on a model 450 Fluidics station.
7. Scanning is performed on an Affymetrix model 3000 scanner with an autoloader.
8. Chip library files specific to each array and necessary for scan interpretation are
stored on the computer workstation controlling the scanner.

3.6. Real-Time Quantitative PCR

Quantification of differentially expressed genes detected by DNA micro-
arrays can be confirmed by real-time PCR. RNA is prepared from each sample
of tissue or purified cells and analyzed by real-time PCR.
1. Briefly, isolated RNA is reverse transcribed using SuperScript II RNase Reverse
Transcriptase (Gibco, Carlsbad, CA).
2. All primer pairs are designed with the Primer Express software (Applied
Biosystems).
3. During primer testing, nontemplate controls and dissociation curves are used to
detect primer-dimer conformation and nonspecific amplification.
4. Direct detection of the PCR product is monitored by fluorescence of SYBR Green
induced by binding to double-stranded DNA.
5. Reactions are performed in a MicroAmp Optical 96-well reaction plate (Applied
Biosystems) using each primer pair in 5 µL of cDNA mix, 5 µL of primer, and
10 µL of SYBR Green Master Mix (Applied Biosystems) per well.
6. The gene-specific PCR products are continuously measured by means of the
GeneAmp 5700 Sequence Detection System (Applied Biosystems) during
40 cycles (see Note 4).
7. The threshold cycle (which equals the PCR cycle at which an increase in reporter
fluorescence first exceeds a baseline signal) of each target product is determined
and normalized to the amplification plot of GAPDH.
8. All experiments are run in triplicate, and the thermal cycling parameters are main-
tained at constant values.
9. Fold change is calculated relative to control cycle threshold (CT). The CT
value is defined as the number of PCR cycles required for the fluorescence
signal to exceed the detection threshold value. With a PCR efficiency of 100%,
the C T values of two separate genes can be compared (∆C T); the fold differ-
ence = 2–(CT – CT control).

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 Microarray Analysis of Gene Expression 7

3.7. Bioinformatics
3.7.1. Data Management
1. For each microarray, we store the expression level of approx 45,000 probe sets
linked with the experimental group as the class label.
2. Experimental data are catalogued in a manner consistent with the Minimum
Information About a Microarray Experiment (MIAME) checklist published by
the Microarray Gene Expression Data Society (MGED) (8). Documentation
should include the experimental design, samples used, sample preparation and
labeling, hybridization procedures and parameters, measurement data and speci-
fications, and array design as described in the checklist on the MGED website
(www.mged.org/Workgroups/MIAME/miame_checklist.html).
3. All data are available upon request. All samples from experiments are assigned a
sequential number with each individual aliquot/sample given an extension of this
number. Therefore, each aliquot/sample can be individually tracked and linked
with our microarray data. Experimental data including date, mouse strains, date
of birth, weight, and sex for both the donor and recipient, as well as time of
harvest, are stored for each experiment.
4. All microarray data including .dat files should be backed up and accessible by
password-protected Internet access.

3.7.2. Low-Level Data Processing

1. Raw microarray data are normalized and processed by the Affymetrix Microarray
Data Analysis Suite (MAS5.0), GCRMA, or dChip software.
2. The quantitative RNA level is computed from the signal strength of the 11 pairs
of perfect match (PM) and mismatch (MM) probe pairs representing each gene,
where MM probes act as specificity controls that allow the direct subtraction of
background and cross-hybridization signals.
3. In the analysis with MAS5, each array is normalized to a standard of 2500 units
per probe set. To determine the quantitative RNA level, the averages of the dif-
ferences (avg diff) representing PM – MM for each gene-specific probe set are
calculated. The expression of each probe set is categorized as present (P), mar-
ginal (M), or absent (A).
4. We also tried to use the rank invariant set normalization and model-based expres-
sion algorithm by dChip software as well as the GCRMA algorithm implemented
in BioConductor (http://www.bioconductor.org/) to perform the normalization
and calculate the expression levels. As previously reported, our comparison of
dChip and GCRMA showed that GCRMA identified a greater number of signifi-
cantly modulated genes.
5. To eliminate noise and facilitate future gene selection procedures, a filtering pro-
cess based on a coefficient of variation (CV) is applied to the whole data set of
45,000 probe sets. Nondifferentially expressed genes with a low CV and
nonexpressing genes with low expression levels across all the microarrays are
considered noninformative and are excluded in the subsequent analyses. The class

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 8 Liang, Lu, and Perkins

labels are masked during the gene filtering step to prevent bias in the gene selec-
tion process.

3.8. Algorithms to Cluster Gene Expression Profiles

There are multiple algorithms and software that perform clustering analysis
of gene expression data. Two examples that we have used in our laboratory are
hierarchical clustering and self-organizing maps (SOMs).
3.8.1. Hierarchical Clustering Dendrograms
Hierarchical clustering analysis can be performed using either commercial
or free access software, such as Cluster and TreeView (9) (courtesy of M. Eisen,
Lawrence Livermore Radiation Laboratory, Berkeley, CA) and GeneCluster
(courtesy of Whitehead Institute for Biomedical Institute, Cambridge, MA)
software. Briefly, dissimilarity between groups is determined by calculation of
a difference metric, such as Euclidean distance or the Pearson correlation coef-
ficient, between each series of values.
3.8.2. Self-Organizing Maps
1. SOMs can be generated by GeneCluster among the experimental groups.
2. The number of maps is selected empirically to eliminate clusters with few genes
or large standard deviations. The number of epochs (iterations) of the algorithms
is selected to minimize the standard deviations (SDs) of the groupings and is
limited only by computer time. For example, we commonly used between 100
and 5000 epochs.
3. Using multiple heuristic observations, the goal is to generate maps in which
increased number of nodes produced clusters with low number of genes, whereas
decreased number of nodes produced larger SDs. Also, increasing the number of
epochs (= 500) should not produce substantial changes in the number of clusters
or SDs.

3.9. Statistics
For analysis of gene expression data between two experimental groups,
the correlation or regression analysis between the response variable and the
expression level is calculated.
1. The expression levels of each gene, under different experimental conditions or
different groups of samples, are compared by the statistical methods just given.
2. The p-values are calculated and adjusted by false discovery rate (FDR) control.
Calculation of the p-value and FDR adjustment is conducted via R, using functions
provided by the packages from BioConductor (http://www.bioconductor.org/).
Genes whose FDR adjusted p-values below a specified level are selected as dif-
ferentially regulated and are used in follow-up studies.

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 Microarray Analysis of Gene Expression 9

3. Alternatively, for the two-class problem, genes can be selected by building a

classification model, e.g., the support vector machine (SVM) model, estimating
the prediction error by cross-validation, and also selecting important genes by
evaluating their relative contribution to the classification model. The RSVM
algorithm was developed by Zhang and Lu (10) and has been used successfully in
protein marker identification problems (http://www.stanford.edu/group/wonglab/
RSVMpage/R-SVM.html).

3.9.1. Selection of Significantly Differentially Expressed Genes

Our previous studies compared the power to detect significantly modulated
genes in duplicate versus quadruplicate microarrays, analyzing independent
samples. These results established that quadruplicates allowed detection of
greater numbers of modulated genes. Based on the number of significantly
regulated genes detected, a cost analysis indicated that the most efficient
approach would be to analyze quadruplicate samples. Therefore, quadruplicate
microarrays are recommended to estimate the individual and replicate varia-
tions, to ensure the maintenance of quality control standards, and to determine
whether the experimental variation exceeds the technical variation for our data.
1. The data can be classified according to the categories of the experimental groups,
and an initial two-way comparison can be performed between groups. In these
studies the expression of each gene in the experimental group is compared with
the corresponding expression samples in the control group.
2. We can identify differentially expressed genes by applying FDR adjustment to
the raw p-values of all genes, and we can select genes that are differentially
expressed, with the FDR controlled below tuned criterion, e.g., 5%.
3. Using this approach, we can select subsets of genes that are significantly differ-
entially expressed in the experimental and control groups.

3.9.2. Randomization Test of the Gene Selection Procedure

1. The gene selection procedure should be validated by a randomization test.
2. The class labels of the microarray are randomly permuted, and the same gene
selection procedure and Gene Ontology (GO) annotation analysis are implemented.
3. This randomization is performed multiple times and the number of genes, as well
as highly concentrated GO terms, if any, is compared with the discoveries based
on true class label.

3.9.3. Statistical Validation

1. The predictive power of a certain subset of genes, with a certain prediction model,
can be estimated by cross-validation.
2. In cross-validation, we will leave a small number of samples out, e.g., leave one
out when the sample size is very small, or leave 10 to 20% out when we have a
moderate number of samples; then we build a predictive model based on the

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 10 Liang, Lu, and Perkins

selected genes of the training set and predict the class labels of the left out
samples.
3. This is equivalent to testing the model using another independent test set, but the
cross-validation procedure is performed multiple times rather than only one split-
ting of the whole data set. In this way, we can get a better estimation of the
predicting error rates.
4. When doing leave-one-out cross-validation, the number of iterations typically is
the same as the sample size, i.e., leave out one sample each time. When doing
cross-validation by leaving out a portion of samples each time, the number of
iterations is no less than the sample size; the only limitation is the computational
power available.

3.10. Biological Interpretation

Microarray technology can rapidly generate large databases of gene expres-
sion profiles. The challenge in array studies is to link the descriptive expression
profiles with relevant biological processes or clinical diagnoses. A common ana-
lytical approach has been to select a few genes with the greatest change in
expression and correlate them with a specific disease or biological phenotype.
However, this approach eliminates >99% of the data from subsequent analysis
and may ignore important biological observations. For example, is a gene
upregulated early, but to a low level, less important (or a less effective thera-
peutic target) than a gene upregulated late, but to a high level? Thus, many
studies have used arbitrary criteria, such as the ratio of expression, to focus on
only a few of the observed changes.

3.10.1. Biological Validation

In addition to the statistical validation of our results, we also assess the bio-
logical interpretation of our selected genes based on determination of the bio-
logical processes, molecular functions, and cellular components, as defined by
the GO database. In association with each gene-based classification, we vali-
date the biological significance of our candidate lists of differentially expressed
genes by GO annotation.
1. The list of genes identified as differentially regulated are analyzed by GO anno-
tations to find highly concentrated biological processes, cellular components, and
molecular functions.
2. The GO analysis is performed by GeneMerge (http://www.oeb.harvard.edu/hartl/
lab/publications/GeneMerge/GeneMerge.html) (11) and GeneNotes (http://
combio.cs.brandeis.edu/GeneNotes) software.
3. The pathways that evolved in the regulated genes are found by matching the gene
list with a pathway database, e.g., the Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathway database.

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 Microarray Analysis of Gene Expression 11

4. Information from other public databases will also be integrated whenever neces-
sary by GeneNotes software. The databases include, but are not limited to, chro-
mosome mapping, gene annotation, homologous genes, unigenes, RefSeq, protein
sequences, protein-protein interaction, and PubMed literature.
5. For regulatory motif finding, we cluster genes by their expression profile using
GeneCluster, GenePattern (http://www.broad.mit.edu/cancer/software/software.
html), and TightCluster (http://www.pitt.edu/~ctseng/research/tightClust_
download.html) (12).
6. Motifs are found from the clustered genes by de novo motif-finding algorithms,
including BioProspector (13), MDscan (14), and CompareProspector (15);
they are validated by the TransFac database.

3.11. Quality Control

1. Various sources of experimental noise will inevitably be introduced into the data
set; thus we must assess the noise before interpreting the data with classification
information.
2. The level of experimental noise should be estimated by replicates of microarrays
on samples that are processed independently (including RNA processing and
microarray hybridization).
3. With this approach, the within-replicate experimental variation level can be esti-
mated and compared with individual variations and between-group variations.
Only when the replicate variation is significantly smaller than the individual
variation and in turn the individual variation is significantly smaller than the
between-group variations, can any differences observed by comparing different
groups be considered significant.
4. Another source of variation is the batch effect. Because of the ongoing experi-
mental design, it is not feasible to collect all samples and perform the microarray
studies simultaneously. Therefore, it is essential to monitor possible batch effects
to avoid/correct any bias between batches.

4. Notes
1. Enzyme digestion methods vary. This modified method contributes to a higher
yield of viable cardiac cells during our previous experiments.
2. Each gate can clearly show the two populations of GFP+ and GFP– cells.
The purity of each isolated cell fraction was >99% by FACS.
3. Every purified cell population should be 10,000 or more to get enough RNA.
During RNA extraction, you may not be able to see the pellet at the final step.
4. Because of the lower concentration of cDNA (≤1.0 µg/µL), we used 50 cycles for
the primer pair amplification to obtain good production, instead of the 40 cycles
we usually use for real-time PCR.

Acknowledgments
We thank Walter Zybko PhD, John Daley, and Suzan Lazo-Kallanian for
technical support.

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 12 Liang, Lu, and Perkins

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transplantation: a gene expression profile analysis. Physiol. Genomics 15, 52–64.
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T-cell receptor gene deficiency in graft rejection by gene expression profiles.
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Exploring the Variety of Random
Documents with Different Content
Pessimism, 295 et seq.

Pfaff, F., 219

Pfleiderer, O., 256, 259, 285

Philip, the Fair, 152

Philosophical Errors, 327

—— Training, great Want of, 321

Philosophy, 7, 16, 21, 28, 36, 44, 78, 242, 275, 292 et seq.

Philosophy and the Faith, 92

—— Scholastic, 49, 53

Piazzi, G., 209

Pindar, 74

Pius IX., 99, 162, 165, 166

Pius X., 99

Plate, L., 206, 237, 240, 241, 243 et seq., 249, 287

Plato, 52, 74, 249, 269, 275, 337

Plutarch, 349

Poggendorff, 209

Pohle, J., 209

Poincaré, H., 114

Pope, his Person, 98

Popes, and the Universities, 150 et seq.

Prantl, K. von, 324

Prayer, 46

Pressensé, F. de, 299

Primordial Genesis, 241

Progress, 159

Promoting the Christian Faith, the Aim of Founders of Universities,

367

Protestantism, 19, 27 et seq., 44, 45, 51, 54, 66, 77, 97, 117, 128,
129, 140, 168, 193, 195, 202, 255, 293, 298, 359, 363, 390, 396

Ptolemy, 5

Pythagoras, 4, 140

Quenstedt, F., 7, 219, 223

Rade, M., 282

Radicalism, 332

Ramsay, W., 7

Ranke, L. von., 116, 179

Ratzel, F., 115

Reason, its Limitations, 7, 14

Reformation, the, 27, 28, 363

Reimarus, H. S., 258

Reinhold, G., 391

Reinke, J., 7, 115, 223

Relative Truth, 157

Religion, 16, 20, 25, 28, 51

—— abandoned, 289 et seq.
—— distinguished from Science, 266
—— of natural Reason, 28, 51

Religious Instruction of Children, 45

[pg 417]

Remus, John, 196

Renan, E., 54, 248, 258

Research, and Faith, 59

—— Definition of, 9

Restraint, proper, of Science, 90

Revelation, 29, 51, 72, 77 et seq., 90, 125, 297

—— Proof of, 138

Revolution, French, 29, 36

—— of 1848, 363

Rheticus (G. Joachim), 195, 201

Rhodius, 140

Riccioli, J., 190

Right of Christians, to be represented, 367

—— to teach, natural, 369

Rights of Teacher, not unrestricted, 346

Ritter, K., 218

Romanes, G., 221

Roscellin, 158

Rosenberger, 118

Rosmini-Serbati, 110

Rothenbücher, 30

Rousseau, J. J., 28

Rudder, P. de, 249

Ruville, A. von, 77

Sabatier, A., 26, 39, 78, 262

Saint-Hilaire, 223

Saitschick, 392

Sarcey, 173

Savigny, F. von, 355

Scepticism, 35, 47, 55, 293

Schafhäutl, K. von, 219

Scheiner, Ch., 125, 191

Schell, H., 136, 172

Schelling, 4

Scherr, J., 38
Schiaparelli, G., 191

Schiller, 274

Schleiermacher, 45, 54, 290

Schmiedel, P., 282

Schneider, W., 116

Schönbein, 7, 218

Schönberg, Cardinal, 194, 201

Schools, free, 22

Schopenhauer, 35, 272, 274

Schwann, Th., 220

Schwegler, A., 28

Schweitzer, A., 282

Science, an Activity of the human Mind, 6

—— anti-Christian, its Danger, 329 et seq.
—— Definition, 3 et seq.
—— Errors of, 115 et seq.
—— grave Charges against Modern, 329
—— Limitations, 7
—— Power of, 322
—— restricted by accidental Conditions, 361
—— subject to God, 6
—— subject to Imperfections of human Mind, 6, 31
—— subject to Truth, 6
—— Vocation of, 279

Sciences, profane and the Faith, 88

Scientific Research, Methods, 158
—— Teaching, Definition, 316

Scientists, Catholic, 384 et seq.

Scripture, does not teach profane Sciences, 84

—— Interpretation, 27
—— Narratives not to be taken in literal Sense, 82 et seq.

Secchi, A., 191, 208

Sedgwick, A., 219

Seminaries, 400

Sensuality, Emancipation of, Danger to Civilization, 356

Sexual Perversities, 347

—— Practice, natural, 346
—— Questions, 325
—— Reform, 347

Sham-Science, 313

Silence not Denial, 11

Smet, de, 86

Smith, Adam, 28

Smolko, S. von, 359

Socialism, 111, 349, 350

Socialists, 331

Social question, 30

Sociology, 30

Socrates, 7

Soul, the, 46 et seq.

—— the, an illusion, 288 et seq.

Spencer, H., 243, 313

Spicker, G., 22, 26, 292, 324

Spinoza, B., 41, 179

Stägemann, 36

[pg 418]

State, the, and Freedom of Teaching, 340 et seq.

Steudel, 236

Strauss, D. F., 54, 65, 240, 258, 267, 273, 280, 283, 286, 287, 315,
364

Stütz, 119

Subjectivism, 33 et seq.

Supernatural, Factors to be excluded, 235 et seq.

—— the, inadmissible, 31

Supervision of Teaching, Ecclesiastical, 389

Sybel, L. von, 246, 292

Syllabus, the, 55, 94, 115, 162 et seq.

Tanner, A., 192

Targioni-Tozzetti, 194

Teachers, anti-Christian, 358

—— Catholic, small Number of, 365
—— Jewish, 365

Teaching, Definition of, 10

—— of the Church, as distinguished from Opinions of Theologians,
82 et seq.

Tews, J., 358

Thénard, L., 217

Theocentric View of the World, 19

Theologians, Catholic, of Repute, 384 et seq.

Theological Literature, Catholic, 384 et seq.

Theology and Progress, 381 et seq.

—— a Science, 378 et seq.
—— History of, 403

Theophobia of Science, 234, 241

Theory of Rights, individualistic, 313

Thomas, St., 82, 84, 155, 262, 353, 388

Thomasius, Chr., 344, 363

Thomson (Lord Kelvin), 74, 238, 249, 251 et seq., 381

Toland, J., 28

Treitschke, H. von, 129, 179

Tröltsch, E., 28, 134, 167, 298, 356, 389

Truth, relative, 49 et seq.

Tyndall, J., 217, 224

Überweg, F., 267

Uhlich, L., 291

United States, 111, 368

Universities, 150, 341 et seq.

—— and the Church, 371
—— Catholic, 370
—— free, 368

University, and Theology, 398

—— Teachers, 17
—— vanishing Respect for, 334

Unprepossession in Research, 121 et seq., 357

Urban IV., 155

Urban V., 151

Urban VIII., 96, 186, 189

Vaillant, Anarchist, 350

Valerius, Maximus, 319

Varnhagen, 36

Vatican Archives, 95

Vatican Council, 68 et seq., 90, 103, 106, 109, 130

Vaudin, 119

Vierort, K. von, 220

View of life, Christian, 252

—— of the World, anthropocentric, 19
—— —— Christian, 14, 27
—— —— humanitarian, 18, 21 et seq., 31
—— —— theocentric, 19

Views of the World, various, 13, 22, 159, 294

Vigilius, St., 180 et seq.

Vincent, St. of Lerin, 383

Virchow, R. von, 116, 224, 241, 323

Vogt, K., 30, 115, 224

Volkmann, A., 220, 223

Volta, A., 212 et seq., 224

Voltaire, 28, 326

Vries, H. de, 220

Waagen, W., 223

Wahrmund, L., 86

Wallace, A., 119

Walsh, J. J., 208

Walther, W., 284

Washington, George, 349

Wasmann, E., 116, 223, 243, 249

Wehner, von, 405

Weismann, 242

[pg 419]

Weizsäcker, 257

Westermark, 50

Westhoff, 177, 74, 145, 276, 282

Wimpheling, 156

Wobbermin, G., 245

Wolf, R., 207

Wöllner, Minister, 363

Wundt, W., 24, 52, 62, 71, 137, 235, 243, 254, 288, 290

Young, Th., 119

Zacharias, Pope, 180

Zedlitz, von, 363

Zeller, E., 246, 255

Ziegler, Th., 59

Zittel, 116

Zöckler, 7, 181, 201

Zoen, Bishop, 151

Zola, 248 et seq.

Footnotes

1. Whenever we use here the word “modern,” we do not take

it in the sense of “present,”—the Christian view of the world
is also a present one, and is still of the utmost importance,
—but in the sense of “new” in contrast to the time-
honoured and inherited.
2. The difference between the Protestant and the Catholic
manner of reasoning is stated by the convert, Prof. A. von
Ruville, as follows:

“My mind had harboured up to now the characteristically

Protestant thought that I, from my superior mental
standpoint, was going to probe the Catholic Church, that I
was going to pass an infallible judgment on her truth or
untruth, and this in spite of my being ready to acknowledge
the truth in her. But now I became more and more
conscious of the fact that it was the Church who had a right
to pass judgment on me, that I had to bow to her opinion,
that she immeasurably surpassed me in wisdom. Many
details, which I was inclined to criticize, demonstrated this
to me, for in every instance I recognized that it was my
understanding that was at fault, and that what appeared to
me as an imperfection was rooted in the deepest truth. In
 this way I was gradually brought to the real Catholic
standpoint, to accept the doctrines immediately as Truth,
because they proceeded from the Church, and then to
endeavour to understand them thoroughly, and to reap
from them the fullest possible harvest of Truth. Formerly,
with regard to Protestant doctrines, I always retained my
independence and the sovereignty of my judgment. Why
should I not have had my own opinion, when every
denomination and every theologian had an individual
opinion? How different with the Catholic Church. Before her
sublime, never varying wisdom, as it is proclaimed by every
simple priest, I bowed my knees in humility. Compared to
her experience of two thousand years my ephemeral
knowledge was a mere nothing” (Back to Holy Church, by
Dr. Albert von Ruville, pp. 30, 31).
3. Infallible teachings are often also called dogmas. But they
are not always dogmas in the strict sense. In the strict
sense dogmas are such truths as are contained in divine
revelation, and are proclaimed by the infallible teaching
authority of the Church to be believed as such by the
faithful. In a broader sense those tenets are often called
dogmas which are presented by revelation or by the Church
as infallible truths. In this sense all teachings of faith clearly
found in Holy Scripture are dogmas, even if not declared by
the Church. In this sense Protestants, too, believe in
revealed dogmas.
4. “They that have received the faith through the ministry of
the Church can never have just cause for changing their
faith or calling it into doubt” (Sess. III, ch. 3). The Vatican
Council did not thereby mean to say that an exceptional
case could not happen where some one, without fault of his
own, might fall away from his faith, either on account of
insufficient religious instruction, or of natural dullness or
exceptional misfortunes in the circumstances of life in which
he may be placed. The theologians who worded the
 decision also say that the Council did not intend to
condemn the opinion expressed by many older theologians,
that under certain conditions an uneducated Catholic might
be led in such way into error as to join another faith
without committing a sin. (cf. Granderath, Const. Dog. ss.
oec. Concl. Vat. 69).
5. At a certain Austrian university, where the custom obtains
that a member of a faculty of the university, in the regular
order of the faculties, publishes during the year a book on
some study in its particular branch, the turn came to the
theological faculty. One of its members then issued a work
on moral theology, of course with the ecclesiastical
Imprimatur. Upon this being discovered the senate resolved
not to acknowledge the book as a university publication,
nor to issue it as such, as is usually the custom. They
believed they saw in the Imprimatur a degradation of
science and a violation of its freedom—a procedure entirely
in accord with the traditional narrow-mindedness and
intolerance of liberalism.
6. A clear understanding of the case of Galileo has been made
possible only since the year 1877, when the papers of the
trial were published by two men of opposite religious views,
—the Catholic-minded historian, de l'Epinois, and the liberal
author, K. Gebler, who in 1876 had already published a
work on “Galileo Galilei and the Roman Curia,” in the spirit
of the anti-clerical tendency of the times. Yet, in spite of his
attitude, he was given free permission to copy the papers—
a magnanimity by which the Holy See has earned the
gratitude and admiration of every fair-minded lover of
history. In more recent times, A. Favaro published, in 1890-
1907, a work of twenty volumes containing all the papers
relating to the trial of Galileo, “Opere di Galileo Galilei,
Edizione Nazionale.” He, too, had access to the
ecclesiastical archives, which he acknowledges with thanks.
 It may be said now that the Galileo case has been settled
by documentary evidence.
7. After visiting Thomson at Kreuznach, Helmholtz wrote: “He
surpasses all great scientists I have personally met, in
acumen, clearness and activity of spirit, so that I felt
somewhat dull beside him.” Helmholtz himself (died 1894)
has never expressed himself about religion. Absorbed by his
scientific work, he seemed to have been indifferent to
religion, but according to his biographer his father was a
decided theist, and his philosophical views were held in
great esteem, and partly subscribed to, by the son.
According to Dennert, Helmholtz attended church now and
then, and even partook of holy communion. Of decided
religious bent of mind was Helmholtz's fellow-countryman,
and co-discoverer of the law of energy, Robert Mayer. At
the Congress of scientists at Innsbruck, in 1869, Mayer
ended his address with the significant words: “Let me in
conclusion declare from the bottom of my heart that true
philosophy cannot and must not be anything else but
propædeutics of the Christian religion.” His letters breathe
piety. For a time he had the intention of joining the Catholic
Church.
8. Others take refuge in the fantastic theory of an “All-
Animation.” According to it all organisms, including trees,
shrubs, grasses, are possessed of a soulful sensation and
feeling for the purposes they serve, and for the elaborate
actions they undertake: this is the reason for their efficacy,
not because a wise Creator had arranged them thus. R. H.
Francé exclaims triumphantly: “When the powers that be
should ask in their dissatisfaction: ‘Where has God a place
in your system?’ we can answer calmly: ‘We do not need
the hypothesis of a personal God.’ ” God is superfluous—this
is the precious gain which this unscientific explanation is to
yield.
 9. Compare Corpus Inscriptionum Latinarum XI (1883, vii.).
10. L. M. Hartmann, Theodor Mommsen (1908), 81. The author
of the biography is a Jew. There is a much-circulated story,
alleged to come from F. X. Kraus. Mommsen is said to have
told Kraus, inasmuch as neither the origin, nor nature, nor
the spread of Christianity can be explained by natural
causes, and since he, in his capacity of historian, could
never acknowledge anything supernatural, therefore the
fourth volume will remain unwritten.
11. Nietzsche, “Thus spoke Zarathustra.”
12. “Veritati ut possetis acquiescere, humilitate opus erat, quae
civitati, vestrae difficillime persuaderi potest” (De civit. Dei,
X, 29).
13. Plato, Phil. 6 c. Similarly Pythagoras, Aristotle, and Cicero.
14. Dial. c. Tryph. 2.
15. “But for the retention of names and terms Harnack leaves
nothing of the specific nature of Christianity,” admits the
Protestant Professor of Theology, W. Walther, in his book,
“Harnack's Wesen des Christentums” (1901).
16. Uhlich, founder of a community of free-thinkers, who died
in 1873, thus describes his evolution from rationalism to
atheism: “At the beginning I could say: We hold fast to
Jesus, to Him who stood too high to be called a mere man.
Ten years later I could say: God, virtue, immortality—these
three are the eternal foundation of religion. And after ten
more years I could issue a declaration wherein God was
mentioned no more.” Similar progress in spiritual
disintegration has been shown by Liberalism in recent
years: first it partially abandoned Christian dogma, without
however quite breaking loose from it; in the eighteenth
century rationalistic enlightenment tore loose from all
 revelation, adhering only to natural religion: to-day even
this is lost.
17. Dr. Spencer Jones, an Episcopal clergyman, says in his
book, “England and the Holy See”: “For the Episcopal
Church the junction with Rome, with its sharply defined
dogmas, its supreme ministry, and its firm leadership, is a
question of life. More and more the supernatural belief is
replaced by individual opinions, a condition which in itself
causes faith to disappear. A condition like the present,
making it possible that in one and the same congregation
the most pronounced contrariety of opinions in respect to
most essential tenets, as well as a general confusion of
minds, is not only tolerated, but directly welcomed, such a
condition cannot endure in the long run.”
18. A French author, G. Goyau, states with truth: “What makes
the (Catholic) Church lovable in the eyes of thinking minds
outside of the Church, is just her uncompromising attitude.
They see a Church steadfast, permanent, imperturbable.
The stumbling block of yore has become for them an isle of
safety. They are thankful to Rome for holding before their
eyes the Christianity, instead of giving them the choice of
several kinds of Christianity, including kinds still unknown,
which they undoubtedly themselves may discover, if so
inclined. They welcome the Roman Church as the ‘Teacher
of Faith’ and ‘Conqueror of Errors,’ and, to quote more of
the forcible language of the Protestant de Pressensé: ‘they
are disgusted with a Christianity for the lowest bidder, but
are impressed by the rigid inflexibility of Catholicism....’ ”
(Autour du Catholicisme social. I. 1896).
19. “The Independent” (New York) of Feb. 2, 1914, reports
under the head freedom of teaching the dismissal of a
professor from the Presbyterian University at Easton, Pa.
After quoting from the charter article VIII, which provides
“that persons of every religious denomination shall be
 capable of being elected Trustees, nor shall any person,
either as principal, professor, tutor or pupil be refused
admittance into said college, or denied any of the
privileges, immunities or advantages thereof, for or on
account of his sentiments in matters of religion,” the report
goes on to say: “it appears however, from the investigations
of the committee, that President Warfield insists that the
instruction in philosophy and psychology has to be such, as,
in his opinion, accords with the most conservative form of
Presbyterian theology.”
20. Prof. Chr. von Ehrenfels, Sexualethik. Similar passages
might be quoted from numerous other books by college-
professors.
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