Parallel%20human%20genome%20analysis:%20Microarray-based%20expression%20monitoring%20of%201000%20genes

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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

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Schena et. al

• Goals
• 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
• 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

• Photolithography using oligos
• Spotting using cDNAs/ESTs

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Schena et. al
Methods

• 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 gt 95 of human
cDNA elements Comparative expression finds
altered fluorescence in 17 array elements
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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

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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

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Schena et. al, Table 2
Correlation of human gene expression from
microarray analysis is confirmed by RNA blot
analyses
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Schena et. al, Figure 3
Microarrays measureexpression in human
tissuesBone marrow, brain, prostate, and heart
Expression levels in genes correlates with
expression level in tissues
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Advantages
Schena et. al

• 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

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Disadvantages
Schena et. al

• Cost
• Commercial availability of microarrays

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Conclusions
Schena et. al

• Microarrays are useful for gene discovery in the
  absence of sequence information
• 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