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132 gene expression in atherosclerotic plaques
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
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
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
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
