SHAPE Society

1. DNA Microarray
Mehran Haidari

2. DNA RNA
PROTEIN FUNCTION

3. Approaches for Characterizing Differential Gene
Expression
Low-throughput or Single Gene Methods
High-throughput or Large-Scale Methods

4. The Hybridization of Complementary
Strands of DNA/RNA
Is the Underlying Principle of All
Methods of Differential Gene Expression.

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. 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. 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. 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

10. 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

12. 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)

13. 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

14. Analysis of Gene Expression
Monitoring Changes in Genomic
DNA
Gene Discovery, Sequencing and Pathway Analysis
When to use Microarray

15. Analysis of Gene Expression
1- Different tissues or different developmental states
2- Normal or diseased states
3- Exposure to drugs or different physiological conditions

16. 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

17. Detailed Protocols
Stanford University
Albert Einstein College of Medicine
NHGRI
Cold Spring Harbor Laboratory
Collection of Protocols
TIGR Protocols
www.cmgm.stanford.edu/pbrown/
www.sequence.aecom.yu.edu/bioinf/microarray/protocol.html
www.nhgri.gov/DIR/LCG/15K/HTML/protocol.html
www.nucleus.cshl.org/wigler
www.protocol-online.net/molbio/DNA/dna_microarray.html
www.tigr.org/tdb/microarray

18. 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

19. 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

20. 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)

21. 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)

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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)

28. 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

29. 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

30. 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

31. 4,000,000 2,500,000
360,0003,613
1,248
EST number in NCBI

32. 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?

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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

41. 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

42. 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

43. 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

44. 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

45. 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