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Search for Genomic and
Proteomic Risk / Protective
Factors Associated with
Coronary Heart Disease
Xiaohong Wu MD, Mehran H...
Hypothesis
1. We hypothesize that genes expressed on peripheral blood
inflammatory cells, monocytes, are distinct among fo...
Design--Study population
 This study will be an analytic case-
control study and either sex, 40 to 80
years old patients ...
Design– Patient group
1- Heart attack: Blood Samples will be collected 2 weeks after the first time
Myocardial infarction,...
Design--Risk Factor
We classify the key risk factors include:
 1. Gender and Age;
 2. Hyperlipidemia;
 3. Hypertension;...
Design--Baseline information
 50 patients from each group will take
part in the study.
 Questionnaire will be filled out...
Experiment Procedure
5ml blood with
anticoagulants
5ml bloodwithoutanticoagulants
10ml blood
sample
Serum
Freeze -80 0
C
B...
Monocytes Isolation and mRNA
Extraction
Blood sample
Monocytes
Total RNA
RNA measured
at 260nm and 280n
( 260/280 ratio 1....
The Process of Gene expression assay
Cells
Poly-A
RNA
AAAA
cDNA
L L L
L
10% Biotin-labeled cRNA
L
Fragment (heat, Mg2+
)
L...
HuSNP Mapping Process
Genomic
DNA
Multiplex
amplification
Labeling
amplification
hybridization scanningWash and staining
(...
Hybridization and staining
L
L
GeneChip
Biotin
Labeled cRNA
+ L
L
L
L
L
L
L
L
L
L
+
SAPE
Streptavidin-
phycoerythrin
Hybri...
Cost for gene expression
arrays assays
U133A $12000/30 Arrays
U133B $12000/30
Arrays
$800 /Patient
SuperScript
Choice Syst...
Cost for HuSNP mapping
assays
HuSNP $1625/5 Arrays
$325 /Patient
HuSNP Reagent
Kit
$150 /patient
Total Cost ~ $700/patient...
Real-Time PCR
 The expressed genes that have more
than 2 fold changes detected by
Microarray will be further verified by
...
Proteomics Analysis
 With the 2-D-gel system proteins are separated by
molecular weight and isoelectric point.
 The gel ...
Process of Proteomics
Analysis
Blood serum protein
preparation
Comparison with

public databanks
Database Development
 We will cooperate with Dr.Fofanov and Dr.
Christoph F. Eick from UH Department of
Computer Science ...
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Project introduction

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Project introduction

  1. 1. Search for Genomic and Proteomic Risk / Protective Factors Associated with Coronary Heart Disease Xiaohong Wu MD, Mehran Haidari MD, Silvio Litovsky MD, Ward Casscells MD, James T Willerson MD, and Morteza Naghavi MD.
  2. 2. Hypothesis 1. We hypothesize that genes expressed on peripheral blood inflammatory cells, monocytes, are distinct among following groups: 1)first time heart attack patients 40-70y(with or without traditional risk factors); 2)people who over 70 with 2 or more risk factors but with no history of heart attack; 3)apparently healthy people without cardio vascular disease or risk factors; 2. We hypothesize that systemic proteomics study of blood serum from different group of patients will identify additional candidate markers, hence result in a much greater capacity to determine individual risk profiles.
  3. 3. Design--Study population  This study will be an analytic case- control study and either sex, 40 to 80 years old patients will be recruited in the study.
  4. 4. Design– Patient group 1- Heart attack: Blood Samples will be collected 2 weeks after the first time Myocardial infarction, acute coronary syndromes from outpatients at Memorial Herman and St. Lukes hospital 2- Suvivors: elderly people w/ 2 or more risk factors but no history of heart attack. We will recruited this group of patients from houston nursing home. 3- Normal: apparently healthy people without cardio vascular disease or risk factors from outpatients at Memorial Herman and St. Lukes hospital with other specialty problem. Gene profile
  5. 5. Design--Risk Factor We classify the key risk factors include:  1. Gender and Age;  2. Hyperlipidemia;  3. Hypertension;  4. Smoking;  5. Diabetes mellitus;  6. Family History;
  6. 6. Design--Baseline information  50 patients from each group will take part in the study.  Questionnaire will be filled out for acquiring baseline information
  7. 7. Experiment Procedure 5ml blood with anticoagulants 5ml bloodwithoutanticoagulants 10ml blood sample Serum Freeze -80 0 C Biochemical profile Monocytes mRNA Rest of the inflammatory Cells store with RNA later (Ambion) @-80 0 C
  8. 8. Monocytes Isolation and mRNA Extraction Blood sample Monocytes Total RNA RNA measured at 260nm and 280n ( 260/280 ratio 1.8-2.0 as high quality) 20ug of RNA for downward Microarray Assay CD14 magneticbeads RNeasy Mini kit(Qiagen) MessageAMP kit(Ambion)
  9. 9. The Process of Gene expression assay Cells Poly-A RNA AAAA cDNA L L L L 10% Biotin-labeled cRNA L Fragment (heat, Mg2+ ) Labeled fragments Hybridize Wash/stain Scan L (Transcript labeling Kit: ENZo Diag) Streptavidin Phycoerythrin Biotin-Labeled Antistreptavidin phycoerythrin Superscript cDNA Synthesis kit
  10. 10. HuSNP Mapping Process Genomic DNA Multiplex amplification Labeling amplification hybridization scanningWash and staining (120ng sample DNA) (1:1000 dilution) (Pool, concentrate) HuSNP reagent kit (affymetrix)
  11. 11. Hybridization and staining L L GeneChip Biotin Labeled cRNA + L L L L L L L L L L + SAPE Streptavidin- phycoerythrin Hybridized Array
  12. 12. Cost for gene expression arrays assays U133A $12000/30 Arrays U133B $12000/30 Arrays $800 /Patient SuperScript Choice System for CDNA Synthesis $688 /kit 25 reaction Enzo Labeling kit $720 /kit 10 reaction $56 /patient $144 /patient mRNA extraction and Misc. Buffer $200/ patient Total Cost ~ $1300/patient
  13. 13. Cost for HuSNP mapping assays HuSNP $1625/5 Arrays $325 /Patient HuSNP Reagent Kit $150 /patient Total Cost ~ $700/patient gDNA Extraction Kit and misc. buffer. 200/patient $3750 /kit 25 reaction
  14. 14. Real-Time PCR  The expressed genes that have more than 2 fold changes detected by Microarray will be further verified by Real-Time PCR
  15. 15. Proteomics Analysis  With the 2-D-gel system proteins are separated by molecular weight and isoelectric point.  The gel is silver stained and comparison of 2-D gels of different materials will indicate protein spots which differ in identity and/or quantity.  Interesting proteins ("differentials") can be characterized further after analyzing protein digests by mass spectrometry.  Comparison with public databanks can be done to reveal the identity and functions of these proteins.
  16. 16. Process of Proteomics Analysis Blood serum protein preparation Comparison with  public databanks
  17. 17. Database Development  We will cooperate with Dr.Fofanov and Dr. Christoph F. Eick from UH Department of Computer Science for developing software and database for further analyzing the gene expression profile from different group of patients in order to gain further genetic information that would help us to be able to develop new diagnostic and therapies for Coronary Heart Disease.

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