The document describes a microarray study to analyze gene expression in atherosclerotic plaques and correlate it with factors related to plaque vulnerability. Specimens will be obtained from human carotid/coronary arteries and atherosclerotic plaques in mouse models. Gene expression will be profiled using microarrays and correlated with histopathology, pH, temperature, spectroscopy and other variables. Plaques from influenza-infected and drug-treated mice will also be analyzed to identify genes associated with plaque rupture. The goal is to better understand plaque vulnerability and identify potential drug targets.

1. 1
Micro-array study for gene
expression in
atherosclerotic plaques and
its correlation with pH,
temperature, spectroscopy,
histopathology,and other
variables of vulnerability
of plaque rupture

2. 2
History
cDNA microarrays have evolved from Southern blots, withcDNA microarrays have evolved from Southern blots, with
clone libraries gridded out on nylon membrane filtersclone libraries gridded out on nylon membrane filters
being an important and still widely used intermediate.being an important and still widely used intermediate.
Things took off with the introduction of non-porous solidThings took off with the introduction of non-porous solid
supports, such as glass - these permitted miniaturization -supports, such as glass - these permitted miniaturization -
and fluorescence based detection. Currently, about 20,000and fluorescence based detection. Currently, about 20,000
cDNAs can be spotted onto a microscope slide.cDNAs can be spotted onto a microscope slide.

3. 3
Applications of DNA array
 Gene expression profilingGene expression profiling
 De novo gene sequencingDe novo gene sequencing
 Gene mutation analysis(SNP)Gene mutation analysis(SNP)
 Gene mapping and genotypingGene mapping and genotyping

4. 4
What is Expression Profiling
o Technique that determines which genes areTechnique that determines which genes are
“turned on” and which genes are “turned off” in“turned on” and which genes are “turned off” in
response to developmental cues, external stimuli,response to developmental cues, external stimuli,
or a variety of stresses. Useful for comparingor a variety of stresses. Useful for comparing
differences in gene expression between normaldifferences in gene expression between normal
and diseased tissues.and diseased tissues.
o Understand general biological/biochemicalUnderstand general biological/biochemical
processesprocesses
o Identify gene targets for drug developmentIdentify gene targets for drug development
o Functional genomicsFunctional genomics

5. 5
Gene Expression
o Pattern of expression of genes in aPattern of expression of genes in a
particular cell is a characteristic of its stateparticular cell is a characteristic of its state
o Expression patterns of many previouslyExpression patterns of many previously
uncharacterized genes may provide possibleuncharacterized genes may provide possible
clues to their functionalities by comparisonclues to their functionalities by comparison
o Can combine with metabolic schemas toCan combine with metabolic schemas to
understand how pathways are changedunderstand how pathways are changed
under varying conditionsunder varying conditions

6. 6
Probe Design
Target
Target
Label
Bind/Wash
Image
Analyze
Typical Microarray Experiment
Oligo
cDNA
Protein
Total RNA
mRNA
cDNA
Amplication
No-amplication
Enzymatic
Chemical
Salt
Temperature
Concentration
Surface Charge
Fluorescence
Background
Light Source

7. 7
 PROBE DESIGNPROBE DESIGN
Target
Target
Gene
Generate
20-25 mers
Fold
Blast
Select
sequences
Probe
Incorrect
Structures
Eliminated
Incorrect
Sequences
Eliminated
Incorrect
Binding
Eliminated

8. 8
Genes involved in stable and
unstable atherosclerotic plaque

9. 9
Il-6
IL-5
Il-4
IL-3
IL-2
Il-1Inetrleukines
HMG-COA reductase
Apo(a)
ABC
Caveolin
Apo-E
Apo-ALipid metabolism
Inhibitor of apoptosis protein-1, stannin
Caspase
C-myc
Bax
Bcl-2
CG12-1
AG8-1
Ca2-1
Fas ligand
p16
p27
P21
P53Apoptosis1

10. 10
MMP-16
MMP-15
MMP-14
MMP -13
MMP-12
MMP-11
MMP-10
MMP-9
MMP-8
MMP-7
MMP-3
MMP-2
MMP-1Matrix metalloprotienases and their inhibitors
IL-18
IL-17
IL-16
IL-15
IL-14
IL-13
IL-12
Il-11
IL-10
IL-9
IL-8
Il-7

11. 11Papain
Fiacin
Chymopapain
Cathepsin B
Caspases
Calpain
BromelainsCysteine Endopeptidase
Dipeptidyl PeptidaseCathepsin
BrinolaseBrinolase
Renin
Pepsin A
HIV Protease
Chymosin
Cathepsin B
CathepsinE
Cathepsin DAspartic Endopeptidase
TIMP-14
TIMP-9
TIMP-3
TIMP-2
TIMP-1
MMP-20
MMP-19
MMP-17

12. 12
thrombin
prothrombin
Methylenetetraydrofolate reductaseThrombosis
Venombin A
Urinary plasminogen Activator
Trypsin
TPA
Thrombin
Subtilisins
Pronase
Plasminogen Activators
Plasmin
Pancreatic elastase
Kallikerins
FactorXIIa
Factor Xa
FactorVIIa
Factor IXa
Enteropeptidase
Endopeptidase K
Complement Factor I
Complement Factor D
Complement Factor B
Chymotrypsin
AcrosinSerine Endopeptidase

13. 13E-Selectin
Angiotensin receptor
VCAM-1
ICAM-1
Cadherin
Antigens CD 31Cell Adhesion molecules
ACE
nNOS
eNOS
iNOS
bradykinin
endothelin
eNO synthaseEndothelial dysfunction(NO)
Glycoprotein IIA/IIB
Coagulation factor V
ADP
serotonin
TX
PGI2
PAI-1
PA
PAF
ICAM
thrombin

14. 14
UCP
LDH-AOther genes
FGF-a
FGF-b
thromboplastin
IGF
PDGF
VEGF
ELC
MCP-4
MIP-beta
MIP-alpha
Rantes
CCr2(receptor)
MCP-1Chemokines
dismutase
NADP oxidaze
NAD oxidazeOxidants/Reductas,e Redox Regulators
PPAR-gamma
PPRA-alphaNuclear factors and receptors
NF-Kappa-B
Integrin-beta
Integrin-alpha
L-Selectin
P-Selectin

15. 15

16. 16

17. 17

18. 18
Folding
 The next step involves folding of the RNAThe next step involves folding of the RNA
structure, and trying to locate the primers onstructure, and trying to locate the primers on
these structurethese structure
 If the position of the primer is free ofIf the position of the primer is free of
excessive hydrogen bonding, repetition ofexcessive hydrogen bonding, repetition of
bases ,we then select it, and repeat this stepbases ,we then select it, and repeat this step
on all the possible structures of RNAon all the possible structures of RNA

19. 19

20. 20
 Ordering of probesOrdering of probes
 Syntesizing mRNASyntesizing mRNA
 Forming cDNAForming cDNA

21. 21Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparing RNA:
RNA ISOLATION
cDNA PRODUCTION
Designing experiments to profile conditions/perturbations/
mutations and carefully controlled growth conditions
RNA yield and purity are determined by system. PolyA isolation is preferable
but total RNA is useable. Two RNA samples are hybridized/chip.
Single strand synthesis or amplification of RNA can be performed.
cDNA production includes incorporation of Aminoallyl-dUTP.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparing RNA:
RNA ISOLATION
cDNA PRODUCTION
Designing experiments to profile conditions/perturbations/
mutations and carefully controlled growth conditions
RNA yield and purity are determined by system. PolyA isolation is preferable
but total RNA is useable. Two RNA samples are hybridized/chip.
Single strand synthesis or amplification of RNA can be performed.
cDNA production includes incorporation of Aminoallyl-dUTP.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparing RNA:
RNA ISOLATION
cDNA PRODUCTION
Designing experiments to obtain conditions necessary to
induce changes in the plaque to cause its rupture
RNA yield and purity are determined by system.
Single strand synthesis or amplification of RNA can be performed.
OBTAINING THE
TARGET TISSUE

22. 22
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
THE PROCESSTHE PROCESS
Building the Chip:
MASSIVE PCR PCR PURIFICATION
and PREPARATION
PREPARING
SLIDES
PRINTING
Preparing RNA:
CELL CULTURE
AND HARVEST
RNA ISOLATION
cDNA PRODUCTION
Hybing the Chip:
POST PROCESSING
ARRAY HYBRIDIZATION
PROBE LABELING
DATA ANALYSIS

23. 23Department of Statistics, University of California, Berkeley, and
Hybing the Chip:
ARRAY HYBRIDIZATION
PROBE LABELING
DATA ANALYSIS
Cy3 and Cy5 RNA samples are simultaneously
hybridized to chip. Hybs are performed for 5-12 hours
and then chips are washed.
Two RNA samples are labelled with Cy3 or
Cy5 monofunctional dyes via a chemical
coupling to AA-dUTP. Samples are purified
using a PCR cleanup kit.
Ratio measurements are determined via
quantification of 532 nm and 635 nm
emission values. Data are uploaded to the
appropriate database where statistical and
other analyses can then be performed.

24. 24Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
cDNA clones
(probes)
PCR product amplification
purification
printing
microarray Hybridise target
to microarray
mRNA target)
excitation
laser 1laser 2
emission
scanning
analysis
overlay images and normalise
0.1nl/spot

25. 25Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Biological
Question
Sample
preparationMicroarray
Life Cycle
Data Analysis
& Modeling
Microarray
Reaction
Microarray
Detection
aken from Schena & Davis

26. 26
Schematic of probe preparation , hybridization,
scanning and immage analysis

27. 27

28. 28
Our Aim
 We intend first to create our own chip based onWe intend first to create our own chip based on
the genes that we selectedthe genes that we selected
 Then we will correlate the expression profiling ofThen we will correlate the expression profiling of
these genes with the temperature, pH,these genes with the temperature, pH,
spectroscopy,and histopathology of atheroscleroticspectroscopy,and histopathology of atherosclerotic
plaquesplaques
 We will also correlate the expression of theseWe will also correlate the expression of these
genes on the influenza infected mice, as well as ongenes on the influenza infected mice, as well as on
mice, whose plaques are prompted to rupture bymice, whose plaques are prompted to rupture by
injecting drugsinjecting drugs

29. 29
Human Carotid artery Specimens
 We will take specimens of human carotid arteriesWe will take specimens of human carotid arteries
from endarterectomy resultsfrom endarterectomy results
 Specimens will be from both symptomatic andSpecimens will be from both symptomatic and
asymptomatic patientsasymptomatic patients
 We will study the correlation betweenWe will study the correlation between
histopathological findings with those of patient’shistopathological findings with those of patient’s
sypmtoms, and also correlate them withsypmtoms, and also correlate them with
differences in pH,temperature, spectroscopy anddifferences in pH,temperature, spectroscopy and
interpret our findings in terms of expression ofinterpret our findings in terms of expression of
genes in both groupsgenes in both groups

30. 30
Human Coronary artery
Specimens
 We will take specimens of human coronaryWe will take specimens of human coronary
arteries from patients undergoing atherectomy orarteries from patients undergoing atherectomy or
angioplasty with distal protectionangioplasty with distal protection
 Specimens will be from both stable and unstableSpecimens will be from both stable and unstable
patientspatients
 We will study the correlation between geneWe will study the correlation between gene
expression profile, histopathological findingsexpression profile, histopathological findings
patient’s clinical presentation etc, .patient’s clinical presentation etc, .

31. 31
Specimens from Influenza
infected apo-e mice
 We will take specimens fromWe will take specimens from
atherosclerotic plaques of influenza infectedatherosclerotic plaques of influenza infected
micemice
 After achieving this goal we will perform micro-After achieving this goal we will perform micro-
array study on these specimens and will try to findarray study on these specimens and will try to find
out which genes are expressed more in theseout which genes are expressed more in these
specimens, and we will compare our results at thespecimens, and we will compare our results at the
histopathology lab which will define the structuralhistopathology lab which will define the structural
details of such vulnerable plaques.details of such vulnerable plaques.

32. 32
Specimens from Atherosclerotic plaque
of apo-e mice prompted to rupture
 We intend to induce rupture of atherosclerotic plaque inWe intend to induce rupture of atherosclerotic plaque in
apo-e mice by injecting several drugs, which we hope willapo-e mice by injecting several drugs, which we hope will
cause rupture of the plaque by altering the hemodynamiccause rupture of the plaque by altering the hemodynamic
system and raising the oxidative stress and alter thesystem and raising the oxidative stress and alter the
composition of the plaque.composition of the plaque.
 After achieving this goal we will perform micro- arrayAfter achieving this goal we will perform micro- array
study on these specimens and will try to find out whichstudy on these specimens and will try to find out which
genes are expressed more in plaques which are ruptured orgenes are expressed more in plaques which are ruptured or
are prone to rupture, by comparing our results at theare prone to rupture, by comparing our results at the
histopathology lab which will define the structural detailshistopathology lab which will define the structural details
of such vulnerable plaques.of such vulnerable plaques.

33. 33Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Carotid
artery
Coronary
artery

34. 34
• Drugs being used to
induce rupture of
atherosclerotic plaque
in apo-e mice

35. 35
Drugs being used
• LNAME
• Adrenalin
• Cocaine
• Xanthine
• Xanthine Oxidase
• Interleukin-1 beta
• Methionine
• Buthionine Sulfoximine

36. 36
If we do not find
anything
Very pleasant, at least
we shalll find
something new