Download Parallel human genome analysis: Microarray

Parallel human genome analysis:
Microarray-based expression
monitoring of 1000 genes
Mark Schena, Dari Shalon, Renu Heller, Andrew
Chai, Patrick O. Brown, and Ronald W. Davis
Schena et. al
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Goals
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To detect the expression of thousands of genes
simultaneously
Gene expression studies – expression patterns of genes
provide clues to function by comparison
 Gene discovery studies
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Use the microarray assay to identify
Known and novel heat shock genes
 Phorbol-ester regulated genes
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Schena et. al
Types of Microarrays
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Photolithography – using oligos
Spotting – using cDNAs/ESTs
Schena et. al
Methods
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Human cDNA from human T mRNA
transformed by the Epstein Barr Virus with 5’
amino acid modification, amplified by PCR, and
arrayed onto silyated microscope slides
Probes labeled with fluorescin and Cy5-dCTP are
hybridized to 1056-element array and scanned
 Verify expression patterns with RNA Blot
 Array elements that display differential expression
patterns are sequenced
 Compare sequence to Informatics databases
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Schena et. al, Figure 1
Monitoring of heat shock response in control
(37o)and treated Jurkat (43o, human T) cells
- Array contains 10
Arabidopsis controls,
1046 human blood
cDNAs
-White box indicates
altered fluorescence:
-Red boxes indicate
activation
-Green boxes indicate
repression
Hybridization signals observed for > 95% of human cDNA elements
Comparative expression finds altered fluorescence in 17 array
elements
Schena et. al, Figure 2
Elemental displays of activated and repressed genes
Fluorescin labeled probes
from (+) heat-shock and (+)
phobol ester cells are
compared to Cy5-labeled
untreated probes
Data is the average of the
ratios from the 2
hybridizations
17 elements have a 2-fold
alteration in fluorescence
Intensity of fluorescnece is
a measure of mRNA
abundance
Schena et. al, Table 1
14/17 clones matched; proximal and distal ends map
to same gene
Hsp90, dnaJ, polyubiquitin, tcp-1 are highly induced
Novel sequences (B7-B9) have 2-fold induction
Schena et. al, Table 2
Correlation of human gene expression from microarray
analysis is confirmed by RNA blot analyses
Schena et. al, Figure 3
Microarrays measure
expression in human tissues
Bone marrow, brain, prostate, and heart
Expression levels in genes correlates with expression level in
tissues
Schena et. al
Advantages
Small hybridization volumes using cDNA provides
specificity not possible with oligo-based arrays
 High array densities
 Incorporation of fluorescence labeling and detection
 High throughput: sequence-based methods require
serial processing
 Rich number of ESTs makes for more powerful
arrays

Schena et. al
Disadvantages
Cost
 Commercial availability of microarrays

Schena et. al
Conclusions
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Microarrays are useful for gene discovery in the
absence of sequence information
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Parallel assays can monitor gene expression for thousands of
genes
Allows high throughput human genome expression and gene
discovery
Allows for rapid mechanistic examination of
hormones, drugs, elicitors, and other small molecules
Potential capacities for patient screening
Sensitivities of microarrays allows for functional
analysis of transcription factors, kinases, growth factors