Transcript Microarrays

Microarray (Gene Expression)
• DNA microarrays is a technology that can be
used to measure changes in expression levels
or to detect SNiPs
• Microarrays differ in fabrication, workings,
accuracy, efficiency, and cost
• Additional factors for microarray experiments
are the experimental design and the methods of
analyzing the data
• Historically, the technique evolved from
southern blotting
• Think microscale for hybridization of many
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genes
DNA microarray
Green Leaf
Can be used to pinpoint all (most) the
differences between gene expression
between two different cell/tissue
types……..in a single experiment.
Senescing Leaf
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Microarrays measures relative abundance of levels
of gene expression
•Isolation of mRNA, which is relatively unstable and
short lived
•mRNA directs the production of cellular proteins
(although protein synthesis and activation are not
regulated solely at the mRNA level in the cell)
•mRNA measurement can be used to estimate cellular
changes in response to external signals or environmental
changes
•Measuring mRNA is therefore widely used to study
gene expression
•The entire genome of an organism can be probed at a single
point in time
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Microarrays use base pairing a process known as hybridization
C always pairs with G, and
A always pairs with T or U
This helps identify unknown DNA sequences
that might be present in a sample.
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www.affymetrix.com
Types of DNA Probes (reporter) on a
microarray chip
cDNA probes
Oligo probes
• cDNAs from high-throughput
sequencing projects are PCRverified then spotted directly
from multiwell plates.
• Sequence ID is done for
probes of interest after
hybridization data are
acquired.
• As in RNA blot experiments,
DNA probes are not
necessarily gene-specific.
• Oligo sequence design is done
before the array is produced
and experimentation is carried
out  DNA probes are genespecific by design.
• DNA probes are synthesized
based on design specifications
either directly on the fixed
surface or prior to spotting onto
the fixed surface.
Long oligo probes
50 -70-mers
Short oligo probes
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25-mers
‘Spotted’ Array Technology – cDNA & Long
Oligo
DNA ‘probes’ are immobilized in grids or ‘arrays’
onto a small platform, commonly microscope slide.
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The Microarray Technologies
Spotted Microarray
Affymetrix GeneChip
cDNAs, clones, or short and long
oligonucleotides deposited onto
glass slides
short oligonucleotides synthesized in situ
onto glass wafers
Each gene (or EST) represented
by its purified PCR product
Each gene represented multiple times - using
16-20 (preferably non-overlapping) 25-mers.
Simultaneous analysis of two
samples (treated vs untreated cells)
provides internal control.
Each oligonucleotide has single-base
mismatch partner for internal control of
hybridization specifity.
relative gene expressions
absolute gene expressions
Each with its own advantages and disadvantages
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Arrays for Molecular Ecology
studies
• Anonymous array = probes used on the
array are random (not based on known
sequences). Useful for cross species
studies
• Dedicated array = probes of genes that
are specific to particular pathway or
phenotype eg. Plant-herbivore interaction)
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Spots and more spots…..
•Software = Imagene for
scoring the spots
•Looking for intensity
differences
•Ratio based analysis
•
http://www.liv.ac.uk/vets_med_images/lmf/microarray_spots_zoomed.jpg 9
Data scoring
•Example of a heat map from microarray data Red =
upregulated and Green = Down regulated genes
•Many ways to interpret the data but one should
always back up suspect genes with other technique
eg. Northerns or Real Time PCR
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Concerns:
• Reproducibility of experiment = $$ hence not done much
• Data is difficult to exchange due to the lack of
standardization in arrays
• Probe that are designed to detect the mRNA of a
particular gene may be relying on genomic EST
information that is incorrectly associated with that gene
• Helps if data is MIAME (Minimum Information About a
Microarray Experiment) compliant
• The MicroArray and Gene Expression Data (MGED)
group is working on the standardization of gene
expression and relevant annotations
• Lots of statistical concerns and it is important to consult
with a good statistician before embarking on microarray
experiment…lots of $$$ involved
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Transcriptome analysis
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Example of
environmental
factor (CO2
ambient vs
elevated) on
gene families
http://www.sciencephoto.com/media/169131/enlarge
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Comparative genomics of
gene expression
Phylogeny of seven spruce species based
upon AFLPs
Microarray experiment design
Species
7 species, 3 individuals for each species
21 hybridizations total
Individuals
Cy3
Cy5
Spruce microarray
21,842 cDNAs
 11,224 have BLAST hits of e-5 or less
 2,402 have BLAST hits of e-50 or less

 (Much
work needs to be done on annotation)
Inference of lineage-specific expression differences
-relative to mean over all species
-”star” phylogeny assumed
Clone
Lineage
a
-0.03
-0.10
0.05
0.05
…
0.02
0.13
-0.20
-0.22
0.05
…
0.09
-0.52
-0.08
-0.15
-0.01
Lineage
b
-0.01
0.07
0.05
-0.44
Lineage
c
0.18
0.09
-0.22
0.02
Lineage
d
-0.24
-0.17
-0.04
-0.02
Lineage
e
-0.12
0.27
0.15
0.06
Lineage
f
0.19
0.17
0.15
-0.17
Lineage
g
0.03
-0.32
-0.13
0.50
Species
variance
0.024
0.043
0.020
0.080
Error
variance
0.001
0.001
0.001
0.002
Species
/total
0.972
0.972
0.971
0.971
chi-square
(6 df)
206.558
206.558
203.785
203.785
-0.08
-0.20
-0.05
0.04
0.02
0.08
0.21
-0.17
0.17
0.06
0.07
0.05
0.02
0.05
-0.20
0.22
-0.32
0.12
0.46
-0.08
-0.17
-0.12
0.19
-0.20
0.09
-0.13
0.26
0.08
-0.30
0.05
0.019
0.048
0.021
0.071
0.011
0.019
0.049
0.022
0.072
0.011
0.497
0.496
0.496
0.496
0.496
5.917
5.917
5.916
5.913
5.909
0.01
0.04
-0.19
0.15
-0.07
-0.01
-0.03
0.30
-0.01
0.07
0.06
-0.14
-0.23
-0.07
0.07
-0.25
0.55
-0.20
0.00
-0.03
0.23
-0.03
-0.30
0.16
-0.01
-0.12
0.13
0.69
-0.07
-0.01
0.023
0.101
0.132
0.013
0.003
0.721
3.128
4.129
0.425
0.088
0.031
0.031
0.031
0.031
0.031
0.195
0.194
0.191
0.190
0.189
Diversifying or “Darwinian” selection
WS0262_L03
WS0047_G13
WS01027_A02
WS00723_G05
…
WS0061_B08
WS0053_E12
WS0085_A17
WS0038_D14
WS0093_M07
…
WS00937_G12
WS01010_O12
WS00722_B02
WS01028_C20
WS01014_I04
No selection
Stabilizing selection
The evolution of gene expression among seven
spruce species: fractions of clones showing
Darwinian selection vs. neutrality vs. stabilizing
selection
All genes
(n=21,334)
8%
42%
50%
Darwinian
Neutral
Stabilizing
Evolution of gene expression in gene families
involved with resistance
All clones
(n=21,334)
All genes
(n=329)
Phenylpropanoid genes
8%
9%
42%
44%
50%
47%
P450 genes
(n=96)
Terpenoid genes
(n=88)
7%
10%
42%
39%
48%
54%
Darwinian
Neutral
Stabilizing