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1
Micro-array study for gene
expression in
atherosclerotic plaques and
its correlation with pH,
temperature, spectroscopy,...
2
History
cDNA microarrays have evolved from Southern blots, withcDNA microarrays have evolved from Southern blots, with
c...
3
Applications of DNA array
 Gene expression profilingGene expression profiling
 De novo gene sequencingDe novo gene seq...
4
What is Expression Profiling
o Technique that determines which genes areTechnique that determines which genes are
“turne...
5
Gene Expression
o Pattern of expression of genes in aPattern of expression of genes in a
particular cell is a characteri...
6
Probe Design
Target
Target
Label
Bind/Wash
Image
Analyze
Typical Microarray Experiment
Oligo
cDNA
Protein
Total RNA
mRNA...
7
 PROBE DESIGNPROBE DESIGN
Target
Target
Gene
Generate
20-25 mers
Fold
Blast
Select
sequences
Probe
Incorrect
Structures...
8
Genes involved in stable and
unstable atherosclerotic plaque
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 o...
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 thei...
11Papain
Fiacin
Chymopapain
Cathepsin B
Caspases
Calpain
BromelainsCysteine Endopeptidase
Dipeptidyl PeptidaseCathepsin
Br...
12
thrombin
prothrombin
Methylenetetraydrofolate reductaseThrombosis
Venombin A
Urinary plasminogen Activator
Trypsin
TPA
...
13E-Selectin
Angiotensin receptor
VCAM-1
ICAM-1
Cadherin
Antigens CD 31Cell Adhesion molecules
ACE
nNOS
eNOS
iNOS
bradykin...
14
UCP
LDH-AOther genes
FGF-a
FGF-b
thromboplastin
IGF
PDGF
VEGF
ELC
MCP-4
MIP-beta
MIP-alpha
Rantes
CCr2(receptor)
MCP-1C...
15
16
17
18
Folding
 The next step involves folding of the RNAThe next step involves folding of the RNA
structure, and trying to l...
19
20
 Ordering of probesOrdering of probes
 Syntesizing mRNASyntesizing mRNA
 Forming cDNAForming cDNA
21Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter an...
22
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter a...
23Department of Statistics, University of California, Berkeley, and
Hybing the Chip:
ARRAY HYBRIDIZATION
PROBE LABELING
DA...
24Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter an...
25Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter an...
26
Schematic of probe preparation , hybridization,
scanning and immage analysis
27
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...
29
Human Carotid artery Specimens
 We will take specimens of human carotid arteriesWe will take specimens of human caroti...
30
Human Coronary artery
Specimens
 We will take specimens of human coronaryWe will take specimens of human coronary
arte...
31
Specimens from Influenza
infected apo-e mice
 We will take specimens fromWe will take specimens from
atherosclerotic p...
32
Specimens from Atherosclerotic plaque
of apo-e mice prompted to rupture
 We intend to induce rupture of atheroscleroti...
33Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter an...
34
• Drugs being used to
induce rupture of
atherosclerotic plaque
in apo-e mice
35
Drugs being used
• LNAME
• Adrenalin
• Cocaine
• Xanthine
• Xanthine Oxidase
• Interleukin-1 beta
• Methionine
• Buthio...
36
If we do not find
anything
Very pleasant, at least
we shalll find
something new
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Micro array study for gene expression in vp

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Micro array study for gene expression in vp

  1. 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. 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. 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. 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. 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. 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. 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. 8 Genes involved in stable and unstable atherosclerotic plaque
  9. 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. 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. 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. 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. 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. 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. 15
  16. 16. 16
  17. 17. 17
  18. 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. 19
  20. 20. 20  Ordering of probesOrdering of probes  Syntesizing mRNASyntesizing mRNA  Forming cDNAForming cDNA
  21. 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. 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. 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. 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. 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. 26 Schematic of probe preparation , hybridization, scanning and immage analysis
  27. 27. 27
  28. 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. 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. 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. 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. 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. 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. 34 • Drugs being used to induce rupture of atherosclerotic plaque in apo-e mice
  35. 35. 35 Drugs being used • LNAME • Adrenalin • Cocaine • Xanthine • Xanthine Oxidase • Interleukin-1 beta • Methionine • Buthionine Sulfoximine
  36. 36. 36 If we do not find anything Very pleasant, at least we shalll find something new

×