DNA microarrays allow for the high-throughput analysis of differential gene expression. They work by hybridizing fluorescently-labeled cDNA from experimental and control RNA samples to a large number of gene sequences spotted on a glass slide. After hybridization, scanned images are analyzed to determine differences in gene expression levels between the two samples. While a powerful tool, microarray results often require confirmation through low-throughput methods like quantitative RT-PCR due to the risk of false positives. Studies have used microarrays to identify genes involved in atherosclerosis, response to oxidized LDL, and effects of shear stress on endothelial cells.

1. DNA Microarray
Mehran Haidari, PhD
Hospital for Sick Children
University of Toronto

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

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

4. Single Gene Methods
Northern Blotting, cumbersome,time-consuming
Nuclease protection, at least 10 fold more sensitive
Quantitative RT-PCR, state of art

5. 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

6. What is DNA Microaray?
Microarray pioneers:
Pat Brown and Schena
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

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

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

9. Monitoring Changes in Genomic DNA
Hybridization to oligonucleotid is sensitive to
detect single-nucleotide mismatches
Single Nucleotide Polymorphisms (SNPs)
High Density Oligonucleotide Array
Cancer cells typically exhibit genomic instability

10. 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

12. 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

13. 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

14. 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

15. Common Approaches for Microarray Fabrication
1-Contact printing (Patrick O Brown,Stanford University)
2- Non-Contact Printing (Pin and Ring, Bubble Jet, Ink Jet)
3- Photolithography (Affymetrix, Oligonucleotide Microarray)
http://www.affymetrix.com/technology/tech_probe.html

16. Contact Printing

17. Non-Contact Printing

18. Photolithography

20. Affymetrix Technology

21. 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

23. 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 need/slide
Laser capture microdissection (LCM) , incorporation of a
PCR step

24. 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)

26. Data Analysis
Seldom more than two replicates of each experiment
Normalization
First step is during scanning, when sensivity 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

27. 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

28. 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

29. 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

30. Quality Control of 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

31. 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 (sensivity)
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

33. C.C Liew,(1994) sequenced 3500 ESTs representing 3100
cDNA from adult human heart(First cardiovascular catalogue of genes)
The number of cardiovascular ESTs increased to 85,000 (1997)
The latest number(2001) is 111,224 cardiovascular ESTs
The largest cardiovascular cDNA microarray constructed (10,368 ESTs)
Cardiovascular ESTs
Choong-Chin Liew, Director, The Cardiovascular Genome Unit,
Harvard Medical School

34. The number of genes encoded by the Human Genome has been
estimated to be ∼ 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 are derived from cardiovscular tissue
ESTs from cardiovascular tissues or cell type
or from diseased specimens remain limited

35. Cardiovascular EST data from most model organisms are almost nonexistent
The construction of cardiovascular gene databases at different stages of
pathology will cast light on the complex genetic mechanisms underlying
disease of cardiovascular system
DNA microarray technology is still in its infancy
DNA microarray in atherosclerosis is not born yet (or at most
is premature)
Premature

36. B.C.G. FABER did the 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 abundance
Circ Rec 2001.89;547-554 University of Mastricht, Netherland

37. SSH
3 ruptured plaques
3 stable plaques
Forward reaction
n=3000
Reverse reaction
n=2000
Macro array
n=500
Sequencing
n=25
RT-PCR analysis
n=3
RNA in situ hybridization
n=1
Immunohistochemistry
n=1
> two fold difference

38. Perilipin was the known gene that upregulated (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
β actin was down regulated in ruptured plaques

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. 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 Boston-Harvard

41. 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
β ACTIN and GAPDH were use as housekeeping gene
J.C.I 2000,105:653-662 Cornell University

42. 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 of the aorta
The most differentially gene was Regulator of G protein Signaling (RGS5)
Northern analysis and in situ hybridization were used to confirm the results
Circulation Research 2000.8.623

43. 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

44. 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
PNAS2001, 98:8955-8960 Rice University

45. 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
Northern Blotting
Nuclease protection
Quantitative RT-PCR

46. Many genes are expressed constitutively and regulation
of their function is at the transnational 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