Transcript Microarray technique and Functional genomics

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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
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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.genechips.com/GeneChips.html)
Application: http://www.dnai.org/d/index.html
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Microarray: high through-put whole
genome approach
48 grids,
with 31k
probes
Each grid contain 650
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
Microarray
or Chip
Probe: oligos/cDNA
(gene templates)
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Target: cDNA
(variables to be detected)
Samples
Hybridization
Analysis of outcome
Graphical and numerical
exploratory data
Data mining: statistical
model of computations
Classification and
discrimination
Functional annotation
Pathway and pattern
analysis
Understanding
genes implicated
in physiological
responses
(phenotype)
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Raw data and processed data
Raw data-images (treatment Cy5
vs. reference Cy3)
Long oligo plotted microarray
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”
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2x overexpressed
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2x underexpressed
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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.html
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Transcriptome characterization of apical and basal
regions of maize root under water deficit conditions
Henry Nguyen’s 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
Growth
ROS and stress
Redox
Cell wall related
Phenylpropanoid
pathway
Drought
Stress
Cellular protection &
adaptation processes
Signal transduction
& gene expression
Root growth
Signaling:
Sensor/recognition
Plant roots play a
vital role in water and
mineral acquisition,
and are essential for
plant growth and
development. Under
conditions of
drought, roots can
adapt to continue
growth while at the
same time producing
and sending early
warning signals to
shoots, which inhibit
plant growth above
ground.
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Experimental design - pairwise comparison
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WW48
Sharp et al. 2004, J Exp. Botany 2004
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WS48
WW24
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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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WS/WW=Cy3/Cy5
WS/WW=Cy5/Cy3
Array experiments
Dye Swap
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
4. Z-score histogram
2. Log channel A intensity vs. log
channel B intensity
5. Box plot
3. R-I
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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/