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DNA Microarray

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Title: DNA Microarray


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
5
High-throughput Methods
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
When to use Microarray
  • Analysis of Gene Expression
  • Monitoring Changes in Genomic DNA
  • Gene Discovery, Sequencing and Pathway Analysis

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
Cancer cells typically exhibit genomic instability
Single Nucleotide Polymorphisms (SNPs)

High Density Oligonucleotide Array
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
11
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12
What to spot
Publicity available clones(IMAGE)
As many known genes as possible
In_house derived (SSH)
Genes that are most relevant
A combination of both approaches
Custom made/purchased libraries
13
Detailed Protocols
Stanford University
www.cmgm.stanford.edu/pbrown/
www.sequence.aecom.yu.edu/bioinf/microarray/protoc
ol.html
Albert Einstein College of Medicine
www.nhgri.gov/DIR/LCG/15K/HTML/protocol.html
NHGRI
Cold Spring Harbor Laboratory
www.nucleus.cshl.org/wigler
Collection of Protocols
www.protocol-online.net/molbio/DNA/dna_microarray.
html
www.tigr.org/tdb/microarray
TIGR Protocols
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
http//www.affymetrix.com/technology/tech_probe.ht
ml
16
Contact Printing
17
Non-Contact Printing
18
Photolithography
19
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20
Affymetrix Technology
21
Micro or Macro
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
22
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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
A single round of transcription is used to
generate a labeled cDNA probe (RT-PCR)
Most microarray utilize two fluorophores, Cyanine3
(Green emission) and Cyanine5 (Red emission)
They have different size and different
ability for incorporation in cDNA
25
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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
How Much is Significant???
Analyzed gene changes are often expressed as a
fold increase either greater than twofold or less
than 0.5 fold (DeRisi)
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 199614457-60
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 (? Actin,
Cyclophilin, GAPDH)
DeRisi used 90 housekeeping genes and found that
changes that were lt0.5 and gt 2.4 were acceptable
? 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
Accuracy and Precision
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
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
32
Choice of an Array System
Spotted Array Vs Oligonucleotide Chips
Homemade System Vs Commercial System
33
Spotted Array
Advantages
Disadvantages
Gene discovery
Clones processing is cumbersome
Optimal size(specific hybridization)
Lower density than chips
Cross hybridization(repetitive sequence)
Available technology
34
Oligonucleotide array
Disadvantages
Advantages
High density chip
Sequence data required
Oligonucleotid selection rules are not
well defined
Consistent and uniform geometry
Not best target for hybridization
Single Nucleotide Polymorphisms
Expensive
No need for maintaining cDNA clones
35
Homemade System
Advantages
Disadvantages
Technology is available (Spotted array)
Design and building the arrays
After the establishment it is a less expensive way
Not system of choice small number is needed
Tracking of clones and print of material can be
cumbersome and error-prone
Unlimited number of array
Most flexible way
Oligonucleotide technology is not available
Complete control by user
36
Commercial system
Disadvantages
Advantages
Uniformity in array quality
Limited flexibility
Q.C is on the manufacturer
Chip cost are high
Access to private clone resources
User is dependent on the manufacturer
Software availability
37
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
unmatched (novel)
2 million human ESTs deposited in GeneBank and
used as substrate for DAN microarray
38
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39
Cardiovascular ESTs
Choong-Chin Liew, Director, The Cardiovascular
Genome Unit, Harvard Medical School
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)
40
Lack of information
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
No cDNA Library for Atherosclerotic plaques
ESTs from cardiovascular tissues or cell type or
from diseased specimens remain limited
Only 5 of total ESTs deposited in GeneBank are
derived from cardiovscular tissue
41
Premature
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)
42
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 (lt 13 copies/cell)
Mammalian mRNA encoding proteins that regular
cellular behavior are expressed at low abundance
Circ Rec 2001.89547-554
University of Mastricht, Netherland
43
SSH 3 ruptured plaques 3 stable plaques
Reverse reaction n2000
Forward reaction n3000
Macro array n500
gt two fold difference
Sequencing n25
RT-PCR analysis n3
RNA in situ hybridization n1
Immunohistochemistry n1
44
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
? actin was down regulated in ruptured plaques
45
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
46
K.j.Haley et al. treated cultured Human aortic
SMC with TNF? 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)
Boston-Harvard
Circulation20001022185-2189
47
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-? and ICAM-1 were also strongly elevated in
the lesion
? ACTIN and GAPDH were use as housekeeping gene
J.C.I 2000,105653-662
Cornell University
48
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
L.D Adams, S.M Schwartz, University of Washington
Circulation Research 2000.8.623
49
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
Orphan nuclear receptors (PPAR?, LXR and RXR)
and ABC1 were among genes which unregulated after
exposure
Real Time RT-PCR was used to confirm the results
J.B.C 200027548, 37324-37332
50
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
Rice University
PNAS2001, 988955-8960
51
Rajeevan et al. estimated that 30 of
microarray results are false-positive
Microarray findings should be confirmed,at
least by one of the low-throughput gene
expression methods
Northern Blotting
Nuclease protection
Quantitative RT-PCR
Rajeevan et al. J Mol Diag 2001-3-26-31
52
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
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