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155 dna microarray


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155 dna microarray

  1. 1. DNA Microarray Mehran Haidari
  3. 3. Approaches for Characterizing Differential Gene Expression Low-throughput or Single Gene Methods High-throughput or Large-Scale Methods
  4. 4. The Hybridization of Complementary Strands of DNA/RNA Is the Underlying Principle of All Methods of Differential Gene Expression.
  5. 5. Single Gene Methods Northern Blotting, cumbersome, time-consuming Nuclease protection, at least 10 fold more sensitive Quantitative RT-PCR, state of the art
  6. 6. High-throughput Methods Serial Analysis of Gene Expression (SAGE) Rapid Analysis of Gene Expression (RAGE) Representational Difference Analysis (RDA) Suppression Subtractive Hybridization (SSH) Differential screening (plus/minus screening) Differential Display (DD) DNA Microarray Comprehensive evaluation 400,000 Northern Blotting
  7. 7. What is DNA Microarray? A large number of genes deposited onto a glass slide (large scale dot blot) The RNA sample is RT with simultaneous incorporation of label, resulting in labeled cDNA. Microarray slides serve as hybridization targets for labeled cDNA Reverse Northern blotting Patrick O Brown Mark Schena
  8. 8. Basic Steps in Performing a DNA Microarray Experiments 1- Processing cDNA clones to generate print-ready material 2-Printing cDNA clones (or oligonucleotide) onto a substrate 3-Sample RNA isolation 4-Preparation of the probe (e.g. cDNA synthesis and labeling, RT reaction) 5- Hybridization of labeled probe DNA to the DNA arrayed on the substrate 6-Image acquisition, image analysis and data analysis
  9. 9. Microarray Fabrication Technologies In Situ Synthesis of Nucleic Acid (Chip ,GeneChip,oligonucleotide array) 15-20 different 25-mer oligonucleotides Exogenous Deposition of cDNA (cDNA, spotted array) Single DNA fragments, greater 0.5 Kb
  10. 10. Common Approaches for Microarray Fabrication 2-Contact printing (Patrick O Brown,Stanford University) 3- Non-Contact Printing (Pin and Ring, Bubble Jet, Ink Jet) 1- Photolithography (Affymetrix, Oligonucleotide Microarray)
  11. 11. What to spot? As many known genes as possible Genes that are most relevant A combination of both approaches Publicity available clones (IMAGE) In_house derived (SSH) Custom made/purchased libraries
  12. 12. Analysis of Gene Expression Monitoring Changes in Genomic DNA Gene Discovery, Sequencing and Pathway Analysis When to use Microarray
  13. 13. Analysis of Gene Expression 1- Different tissues or different developmental states 2- Normal or diseased states 3- Exposure to drugs or different physiological conditions
  14. 14. Monitoring Changes in Genomic DNA Hybridization to oligonucleotide is sensitive in detection of single-nucleotide mismatches Single Nucleotide Polymorphisms (SNPs) High Density Oligonucleotide Array Cancer cells typically exhibit genomic instability
  15. 15. Detailed Protocols Stanford University Albert Einstein College of Medicine NHGRI Cold Spring Harbor Laboratory Collection of Protocols TIGR Protocols
  16. 16. Two basic substrates commonly used for cDNA printing are glass and membrane filters Chemically treated microscope glass slides are the most widely used support Microarray, Microscope Slide,80000 Spots, 10000-20000 Spots Macroarray, Nylon Membrane, 500,-18000 Spots Micro or Macro
  17. 17. RNA Preparation No difference between total RNA or mRNA Type of tissue might have profound effect on extraction process. 100 -200 µg of RNA is needed/slide Laser captured microdissection (LCM) , incorporation of a PCR step
  18. 18. Sample Labeling Most microarray utilize two fluorophores, Cyanine3(Green emission) and Cyanine5 (Red emission) They have different size and different ability for incorporation in cDNA A single round of transcription is used to generate a labeled cDNA probe (RT-PCR)
  19. 19. Data Analysis Normalization First step is during scanning, when sensitivity of detection is adjusted by the laser voltage Gene expression value can be expressed relative to the expression of housekeeping genes In the absence of control genes, normalization to the median microarray value is popular No consensus, ANOVA Clustering (categorizing genes according to their pattern of expression)
  20. 20. Analyzed gene changes are often expressed as a fold increase either greater than twofold or less than 0.5 fold (DeRisi) How Much is Significant??? With a large number of microarrays, small changes can be statistically valid Elcock et al. detected 1.1 fold changes with 95 % confidence interval when each experimental sample was hybridized to seven microarray slides (with two replicate spots for each gene) Derisi et al.Nat Genet 1996:14:457-60
  21. 21. Housekeeping genes These are genes that are expressed constitutively and their level of expression is thought to be stable, regardless of the sample used (β Actin, Cyclophilin, GAPDH) DeRisi used 90 housekeeping genes and found that changes that were <0.5 and > 2.4 were acceptable β Actin is one of the most commonly used housekeeping genes and it has been shown to be downregulated in heat shock experiments In fact, there is an appreciable amount of literature available to suggest that there is no such thing as housekeeping gene
  22. 22. DNA microarray represents a developing technology, there remain substantial obstacles in the design and analysis of these microarray There are no globally accepted rules or standards for performing controlled microarray experiments A good experiments include more control component then the real comparison Accuracy and Precision
  23. 23. Principles of Q.C in DNA Microarray Down-Scaling of an experiment makes it generally sensitive to external and internal fluctuation Replication of each experiments on multiple array Dual labeling, swapping the dyes for control and treated sample Using a large number of controls on every array
  24. 24. Controls mRNA from genes that are not homologous to the organism understudy (Arabidopsis) cDNA from the organism with high, medium and low expression represented on the array (sensitivity) Cold DNA (e.g., calf thymus DNA, yeast tRNA) is added to block nonspecific annealing Spots of DNA from another organism whose mRNA is not represented in the sample (Background) Total genomic DNA or cDNA clones of common contaminant such as E.Coli and yeast are represented in the array to monitor for contamination
  25. 25. Ontario Cancer Institute Spotted Array Advantages Gene discovery Optimal size(specific hybridization) Available technology Disadvantages Clones processing is cumbersome Lower density than chips Cross hybridization(repetitive sequence)
  26. 26. Affymetrix Genechip Biotinylated cRNA is synthesized from cDNA phycoerthrin linked to avidin is used for labeling Each sample hybridized separately Advantages High density chip Consistent and uniform geometry Single Nucleotide Polymorphisms No need for maintaining cDNA clones Disadvantages Sequence data required Oligonucleotid selection rules are not well defined Not best target for hybridization Expensive
  27. 27. Rajeevan et al. estimated that 30% of microarray results are false-positive Rajeevan et al. J Mol Diag 2001-3-26-31 Microarray findings should be confirmed,at least by one of the low-throughput gene expression methods DNA microarray technology is in its infancy Application of microarray in diabetes is not born or at most is premature
  28. 28. Many genes are expressed constitutively and regulation of their function is at the translational or posttranslational (ApoB ,CFTR, TCR) To date, there has been a relatively poor correlation between gene and protein expression. It is likely that global proteome analysis provides a better representation of the phenotype than does gene expression analysis
  29. 29. 4,000,000 2,500,000 360,0003,613 1,248 EST number in NCBI
  30. 30. Mouse Genechip or spotted microarray Systematic evaluation of insulin signaling and dyslipidemia different tissue,time course TZD or other drugs Parallel study with protoemic What can we do?
  31. 31. Expressed Sequence Tag (EST) A Partial DNA squence derived from a cDNA clone enough to identify the transcript which the cDNA was derived 55% of cardiovascular ESTs matches to known genes 25% with other ESTs and 20% remain unmatch(novel) 2 million human ESTs deposited in GeneBank and used as substrate for DAN microarray
  32. 32. C.C Liew,(1994) sequenced 3500 ESTs representing 3100 cDNA from adult human heart(First cadiovascular catalogue of genes) The number of cardiovascular ESTs increased to 85,000 (1997) The latest number(2001) is 111,224 cardiovascular ESTs C.C.Liew:PNAS,1994;91-10645-10649 C.C.Liew: Circulation, 1997;96:4146-4203 C.C.Liew: J Mol Cell Cardiol, 2001,33,1879-1886 The largest cardiovascular cDNA microarray constructed (10,368 ESTs) C.C.Liew. BBRC, 2000,280-964-969 Cadiovascular ESTs
  33. 33. The number of genes encoded by the Human genome has been estimated ∼ 32,000 - 38,000. Between 21,000 - 27,000 genes are expressed in the cardiovascular system Lack of information No cDNA Library for Atherosclerotic plaques Only 5% of total ESTs deposited in GeneBank derived from cardiovscular tissue ESTs from cardiovascular tissues or cell type or from diseased specimens remain limited
  34. 34. Cardiovascular EST data from most model organisms are almost nonexistent The construction of cardiovascular gene databases at different stages of pathalogy cast light on the complex genetic mechanisms underlying disease of cadiovascular system DNA microarray technology is in infancy DNA microarray in atherosclerosis was not born or at least is premature Premature
  35. 35. First study dealing with differential gene expression in whole-mount specimens of rupture plaques using macroarray Suppression Subtractive Hybridization (SSH) technique isolates low abundant sequence that might not be isolated by use of microarray technology Mammalian mRNA population 20% Abundant transcript (1000-12000 copies/cell) 25% Medium abundant (100-1000 copies/cell) % 50 small number copies (< 13 copies/cell) Mammalian mRNA encoding proteins that regular cellular behavior are expressed at low abundence
  36. 36. SSH 3 ruptured plaques 3 stable plaques Forard reaction n=300 Reverse reaction n=200 Macro array n=500 Sequencing n=25 RT-PDR analysis n=3 RNA in situ hybridization n=1 Immunohistochemistry n=1 > two fold difference
  37. 37. Prelipin is unlikely to be the sole marker of rupture The author used only 10% of differentially expressed gene for doing macroarray A large effort at macroarray and then sequencing would have yield more differences An alternative would be to hybridized the subtractand against a large array Other alternative is the isolation of cell type-specific genes (LCM) rather than plaque-type-specific genes
  38. 38. Perilipin was the known gene that unregulated (confirmed by RT-PCR) 8 of 10 ruptured plaques expressed prelipin while expression was absent in 10 stable plaque Prelipin is a protein which present on the surface layer of intracellular lipid droplets in adipocyte and prevent lipolysis They speculated that this will result in increased lipid retention and plaque destabilization β actin was down regulated in ruptured plaques The down regulation of one gene was not confirmed by RT-PCR
  39. 39. K.j.Haley et al. Treated cultured Human aortic SMC with TNFα and used DNA microarray with 8600 genes to monitor gene expression Marked increase in eotaxin confirmed with northern blotting Immunohistochemical analysis demonstrated overexpression of eotaxin and its receptor in the Human atheroma(SMC) Circulation;2000:102:2185-2189 Bostom-Harvard
  40. 40. McCaffrey et al. compared transcript profile of fibrous cap vs adjacent media of 13 patients ,using macroarray (membrane 588 known genes) Early growth response gene(Egr-1) was highly expressed in lesion (confirmed by RT-PCR) Many Erg-1 inducible genes including PDGF , TGF-β and ICAM-1 were also strongly elevated in the lesion Immunocytochemistry indicated that Egr-1 was expressed in SMC β ACTIN and GAPDH were use as houskeeping gene J.C.I 2000,105:653-662 Cornell University
  41. 41. L.D Adams, S.M Schwartz, University of Washington Adams et al. Compared gene expression of media of aorta and vena cava, using cDNA microarray of 4048 known genes 68 genes had consistent elevation in message expression the aorta The most differentially gene was Regulator of G P rotein Signaling (RGS5) Northern analysis and in situ hybridization were used to confirm the results Circulation Research 2000.8.623 Role of Lipid Rafts in AMPA Receptor Trafficking and Synaptic Plasticity Last ten years has been witness of emerging the concept of lipid rafts which has changed and revolutionized the classical two-dimensional "fluid mosaic" model of plasma membrane (Singer & Nicolson 1972). The new plasma membrane model or so called "liquid-ordered" membrane is based on the existence of organized, detergent resistance discrete detergent resistance microdomain of plasma membrane named lipid rafts. Rafts are membrane subdomains, enriched in cholesterol and sphingolipids. These microdomains act as plat forms for conducting a variety of cellular functions, such as vesicular trafficking and signal transduction (recently reviewed by Simons K, Toomre D, Nature Reviews, 2000, 1:31-39; Galbiati F., et al. Cell, 2001, 106:403-411). Recent data supports that manipulation of cellular lipid composition especially cholesterol and fatty acid contents of plasma membrane bilayer disrupt lipid microdomains integrity, which can subsequently modulate signal transduction and membrane trafficking. There are several classical methods to disrupt rafts integrity including cholesterol sequestration (by using antibiotics such as filipin or nistatin; or by using pore-forming agents such as saponin or digitonin), cholesterol depletion (by methyl-β -cyclodextrin), inhibition of cholesterol synthesis (by statins), and perturbation of raft stability (by using exogenous cholesterol, exogenous gangliosides, exogenous polyunsaturated fatty acids). Several important enzymes and signaling proteins such as insulin receptors, PDGF, eNOS, CD36, src-family of tyrosine kinases are localized in lipid rafts (Ref: ). More recently Suzuki et al. (Suzuki T., et al. 2001, Mol. Brain. Res. 89:20-28) reported evidences for localization of AMPA-type glutamate receptors in the dendritic rafts. Glutamate receptors (AMPAs, NMDARs) activities are essential to many neurological functions. Overactivity of these receptors can cause neurological death as a result of excitotoxicity. Excitotoxicity is a key event leading to neuronal injury in stroke patients. Recent evidence supports central role of AMPA receptors in the pathologies caused by brain ischemia. Although the underlying mechanism (s) are not fully understood, modulating AMPA receptors have been shown to be neuroprotoctive. Therefore
  42. 42. R.M Lawn et al. examined the response of macrophages to exposure to oxidized LDL, using microarray containing 10000 Human genes 268 genes were found to be at least twofold regulated Real Time RT-PCR was used to confirm the results Orphan nuclear receptors (PPARγ, LXR and RXR) and ABC1 were among genes which unregulated after exposure J.B.C 2000:275;48, 37324-37332
  43. 43. L.A Mcintire et al. identified 52 genes with altered expression under shear stress Using DNA microarray in primary human umbilical vein endothelial cells Significant increases in mRNA levels for 32 and significant decreases in expression for 20 genes were reported The most enhanced genes were cytocromes P45 1A1 and 1B1 and human prostaglandin transporter Most dramatically decreased were connective tissue growth factor and endotheline-1 Northern blot analysis confirmed the results obtained on microarray PNAS2001, 98:8955-8960 Rice University