Microarray and Gene Expression Profiling

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Microarray and Gene Expression Profiling

• Henry Nguyen
• Wenjing Tao
• University of Missouri

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Gene expression transcription
Gene Transcription DNA ? RNA
Gene Translation RNA ? Protein
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Expression analysis techniques

• Gene expression data is created by process of
  
• Microarray
• RT-PCR
• Differential display
• cDNA AFLP
• SAGE

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Application of microarray

• DNA microarray, or DNA chips are fabricated by
  high-speed robotics, generally on glass but
  sometimes on nylon substrates, for which probes
  with known identity are used to determine
  complementary binding, thus allowing massively
  parallel gene expression and gene discovery
  studies (http//www.gene-chips.com/GeneChips.html)
  
• Application http//www.dnai.org/d/index.html

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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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Kinds of array features
Synthetic oligonucleotides Affymetrix
genechip Long oligo array PCR products from
cloned cDNAs or genomic DNAs cDNA array
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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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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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Gene Expression Analysis Using Microarrays
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Raw data and processed data
Raw data-images (treatment Cy5 vs. reference Cy3)
Red (Cy5) dot overexpressed or
up-regulated Green (Cy3) dot underexpressed or
down-regulated Yellow dot equally expressed Black
represents areas where neither the reference nor
test DNA hybridized to the target DNA. Intensity
- absolute level Red/green - ratio of
expression 2 - 2x overexpressed 0.5 -
2x underexpressed log2( red/green ) - log
ratio 1 2x overexpressed -1 2x
underexpressed
Long oligo plotted microarray
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Microarray experiment standardization

• MIAME (Minimum Information About a Microarray
  Experiment)
• Experimental design,
• Array design
• Samples, hybridisations
• Measurements and controls
• Databases data storage and exchange
• Public repositories
• GEO (NCBI), GeneX (NCGR), ArrayExpress
  (EBI)
• In-house databases
• Proprietary databases
• Gene Logic, NCI, Synergy (NetGenics),
  Genomics Knowledge Platform (Incyte)

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Functional Genomics of Root Growth and Root
Signaling under drought
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http//rootgenomics.missouri.edu/prgc/research.htm
l
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Transcriptome characterization of apical and
basal regions of maize root under water deficit
conditions
Henry Nguyens lab 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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Primary root growth and drought stress
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Experimental design - pairwise comparison

Sharp et al. 2004, J Exp. Botany 2004
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Characteristics of the maize long oligo array

• Maize oligo array was printed at University of
  Arizona as a
• slide pair
• 57,452 70-mer oligos were designed and
  synthesized by
• Qiagen/Operon
• These oligos represent five diverse sequence
  groups
• TIGR Maize Gene Index (26,000 TCs, 20,500
  singletons)
• TIGR Maize Genome Sequencing Project (9700 AZMs,
  80
• of AZMs are transcriptomes)
• Organelles (460 oligos based on chloroplast and
• mitochondria sequences)
• Non redundant repetitive elements (total 800
  oligos, 400
• oligos in both orientations)
• Favorite genes from maize research community
  (300)

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Array experiments
WW48 region 1 vs. WS48 region1
Technical replicates dye swap
CF024887
BM379776
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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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Computation Analysis

• Statistical analysis mixed linear ANOVA model
  (F test) with Benjamini and Hochberg false
  discovery rate (FDR) multiple testing correction
• Real-time PCR verification of microarray data
• Gene annotation, functional category, and
  pathway analysis

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Real time PCR

• Traditional PCR use agarose gels for detection
  of PCR amplification at the final phase or
  end-point of the PCR reaction, results are based
  on size discrimination.
• Real-Time PCR use small amounts of template,
  measuring the kinetics of the reaction in the
  early phases of PCR, detect the accumulation of
  amplicon during the reaction, measure standard
  curve and calculate template copy number

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Real-time PCR verification of microarray data
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Gene ontology analysis to categorize significant
differentially expressed genes in maize root
region 1
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Example of stress related metabolism pathways
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Transcription factors and regulatory elements in
the promoters of genes in the stress response

• Root region 1
• 97 stress related TFs
• 160 genes with these TFs response elements. Some
  of these are with multiple responsive elements
• Gene regulation networks based on transcription
  factors and their regulation targets are being
  studied using real-time PCR in root tissues over
  a time course
• Root region 2 and region2-3
• These TFs and their regulation targets are also
  being detected in root region 2 and region2-3
  data sets.

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References

• Plant Root Genomics Consortium
• http//rootgenomics.missouri.edu/prgc/index.html
• BASE
• http//bioinf2.rnet.missouri.edu/