Microarray and functional genomics

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Microarray and functional genomics

• Wenjing Tao
• University of Missouri

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Microarray high through-put whole genome approach
Each grid contain 650 probes
48 grids, with 31k probes
Microarray is a tool for analyzing gene
expression that consists of a small membrane or
glass slide containing samples of many genes
arranged in a regular pattern
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Microarray terminology

• Feature - an array element
• Probe - a feature corresponding to a defined
  sequence (immobilized on a solid surface in an
  ordered array)
• Target - a pool of nucleic acids of unknown
  sequence

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Microarray provides the opportunities

• Find the genes and assign them functions
• Predict protein structures and functions
• Reconstruct metabolic, signaling, and other
  pathways
• Reconstruct informational networks
• Link genotype to phenotype
• Use genotype/phenotype to predict relevant
  outcome
• Cross- species comparisons

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Kinds of array features
Synthetic oligonucleotides Affymetrix
genechip Long oligo array PCR products
from Cloned cDNAs Genomic DNA
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cDNA oligonucleotide arrays
100-300 ?m spot
20-25 mers
Schulze and Downward, 2001 Nat Cell Biol 3, 190
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cDNA and long oligo array experiment
Hybridization
Scan
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Affymetrix GeneChip
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(No Transcript)
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Fluorescent microarrays are composed of a
combined two false color laser scanned images
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Image file post-processing

• Single slide normalization GenePix Pro 4.1
• Slide-slide and dye-swap comparison TMEV
  MIDAS
• Cross-slides quality evaluation - GeneSpring
  R script for CV filter
• Mixed linear model analysis of Variance to
  identify significant differentially expressed
  genes R or SAS program
• Data Analysis in the Post-Genomic Era (gene
  annotation, ontology and pathway analysis KOG,
  COG, KEGG, TAIR, Onto-Tools, GenMapp
• Data validation qPCR or Northern blot

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Whole genome approaches to biological questions

• Gene expression
• Gene variation
• Gene function

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Functional Genomics of Root Growth and Root
Signaling under drought
NSF-DB1-0211842, PI Henry Nguyen
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http//rootgenomics.missouri.edu/prgc/research.htm
l
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Drought-stress inducible genes and their possible
functions in stress tolerance and response.
Yamaguchi-Shinozaki et al. JIRCAS Working Report,
2002
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Characterize the transcript profiles of apical
and basal regions of the root growth zone under
water deficit condition using maize long
oligonucleitide arrays
Dr. Henry Nguyens lab, Plant Sciences,
University of Missouri
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Objectives

• To identify genes contributing to root growth
  maintenance under water deficit condition
• To determine genes responsible for progressive
  inhibition of root elongation under water-deficit
  condition
• To compare the differential gene expression in
  root region of progressive inhibition of root
  elongation under water stress with the normal
  growth deceleration in well-watered root region

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Pair-wise comparison of maize root segments using
oligo array
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Characterization of the maize long oligo array

• Maize oligo array, printed at the University of
  Arizona, contains 56,311 70-mer oligonucleotide
  probes, including gt30,000 identifiable unique
  maize genes. 16,915 oligoes do not have any
  annotation.
• 70-mer oligonucleotides in conjunction with
  Operon Qiagen based on the TIGR Maize Database

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Slides feature and dye-swap experiment

• WS/WWCy5/Cy3 WS/WWCy3/Cy5

Dye Swap
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Two-color microarray data feature
1. Channel A intensity vs. channel B intensity
2. Log channel A intensity vs. log channel B
intensity
3. R-I
5. Box plot
4. Z-score histogram
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Flip dye consistency checking
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Summary of the evaluation of replicates
(technique biological)

• 50,000 of the 56,311 genes have intensity gt200
  (at least one channel).
• Confidence of dye-swap is gt 96
• 99.9 confidence limit was estimated by testing
  the coefficient of variance (CV) for replicates

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Mixed linear model analysis of two color
microarray data- producing lists of
differentially expressed genes with low false
discovery rates
To obtain accurate and precise estimates of
gene expression values between treatment and
control, analyze gene effects with a simultaneous
consideration of all blocking factors, a linear
mixed ANOVA model is applied There are two
processes
First, global mixed model was applied Log2(singa
l values) treat dye treatdye
tech_reps_effect array_effect (within treatdye
and tech_reps_effect) Second, take residual
values from the first model and then apply this
model for individual gene Residuals treat
dye tech_reps_effects array(within
tech_reps_effects)
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Gene function categorization of significantly
differentially expressed genes
KOG analysis
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Information storage and processing
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Metabolites
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CELLULAR PROCESSES AND SIGNALING
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Summary

• Microarray is a high through-put tool to identify
  novel genes
• We have identified 19 hundred drought response
  and root growth maintenance related genes
• Combining functional analysis we would find
  drought stress tolerance related pathways and
  genes
• This knowledge will lead to novel approaches for
  improving drought tolerance in maize.

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Thank You !