Microarray Technique

Some background

M. Nath
 Outline

„ Introduction

„ Spotting Array Technique

„ GeneChip Technique

„ Data analysis

„ Applications

„ Conclusion
Now
Blind Guess?
Functional Pathway
Microarray
Technique
 Principle

„ Comprehensive functional analysis of

genome

„ Simultaneous analysis of patterns of

gene expression

„ Genome > Transcriptome > Proteome

 Types of Microarray

„ cDNA Array (Brown et. al., 1995)

„ Genomic DNA Array (DeRisi et. al., 1997)

„ Oligonucleotide Array (Morton et. al., 1998)

Spotted Array Technology
 Spotted Array Technology

Library
 Spotted Array Technology

Printing Slides

„ An array of slides is printed

„ Slides can be glass or nylon
Spotted Array Technology
 Spotted Array Technology

Hybridisation of Slides

Slide developer

Up to 48 slides are developed under uniform conditions

 Spotted Array Technology

Scanning
„ Confocal laser scanner is used
„ Two different lasers to read Red and Green dye intensities

Laser Scanner
„ A Graphic image is saved

„ Imaging software reads Red & Green intensity for

each dot applied
 Spotted Array Technology

Results

Green = Active in Sample 1

Red = Active in Sample 2
Yellow = Active in both samples
Black = Active in neither
 GeneChip Technology
Affymetrix chip

„ Oligos of 25 nt long

„ 40 oligos for detection of each gene

„ 11-20 oligos as Perfect Match (PM)

„ 11-20 oligos as Mismatch (MM) at position

13
GeneChip Technology
GeneChip Technology
 Spotted Array Technology

Features Routine

10-20 µg
Starting material total RNA
Probes pair per gene 1

No. of genes / array 10000

 Spotted Array Technology

Laborious
Inexpensive
Moderate specificity
Moderate representation
Low Density
Cannot detect polymorphism
 GeneChip Technology

Features Routine Limit

5 µg total 2 ng total
Starting material RNA RNA
Detection specificity 1:105 1:106
Discrimination of 70-80% 93%
related genes identity identity
Probes pair per gene 20 4

No. of genes / array 12000 40000

 GeneChip Technology

Easy
Expensive
High specificity
High representation
High density
Can detect polymorphism
Data Analysis
 Data Analysis

Scaling
Linearity

Gene intensity Chip 2

Gene intensity Chip 1

 Data Analysis

Scaling

„ Linear and non-linear models

„ Constitutively and constantly

expressed Maintenance gene

Gene intensity Chip 2

„ More genes on chip

Gene intensity Chip 1

 Data Analysis

Outlier

„ Two chips may differ in expression

for same gene

„ If one replicate deviates several

standard deviation from mean,
remove it
 Data Analysis

Absolute measurements

„ AvgDiff
„ Σ ( PMn – MMn ) / N

„ Weighted AvgDiff
„ Σ ( PMn – MMn ) φn / N
 Data Analysis

Fold Change

„ Log2 of ratio of intensities after being

corrected for background

„ E.g. Log2 (Sample / Control) =

Log2 (Red / Green)
„ =1 : unchanged;
„ >1 : upregulated;
„ <1 : downregulated

„ Affymetrix chip (AffyFold)

„ (Sample - Control) / Min (Sample, Control)
 Data Analysis

Significance Test
„ Test of significance
„ t-test with unequal variance
„ ANOVA and F test
„ REML

„ Non-parametric tests
„ Wilcoxon test
„ Mann-Whitney rank sum test

„ Correction for multiple testing

„ Bonferroni correction
 Data Analysis

Cluster Analysis
„ Single array not suitable
„ Functional analysis
„ Co-regulation
„ New gene discovery

„ Samples collected temporally, spatially …

„ Multiple array & Cluster analysis

„ Clustering of similarly behaving genes

„ Genes with similar functions generally

cluster together
 Data Analysis

Cluster Analysis

„ Cluster analysis
„ Hierarchical clustering
„ K-means clustering
„ Self Organising Maps
„ Distance measures
 Data Analysis

Beyond Clustering

„ Discovery of regulatory elements in

promoter region
„ Identifying regulatory networks
„ Time series approach
„ Steady-state approach
„ Neural network technique
„ Selection of genes
„ Gene finding
„ Selection of regions within the genes
„ Selection of PCR primers
„ Selection of unique oligomer probes
 Data Analysis

Software Package

„ Affymetrix Data Mining Tool

„ Affymetrix NetAffx
„ Biomax Gene Expression Analysis Suite
„ GeneData Expressionist
„ Informax Xpression
„ Invitrogen Corp. ResGen Pathways
„ Rosetta Resolver Gene Expression System
„ Silicon Genetics GeneSpring
„ Spotfire
 Applications

„ Analysis of patterns of gene expression

„ Functional relationship between genes
„ Expression in coregulatory gene group

„ Monitoring changes in genomic DNA

„ Cellular pathways affected by mutation
„ Changes in expression profiles of mutants
 Applications

„ Simultaneous detection of many genes

„ Gene discovery

„ Pathway analysis

„ Molecular basis of disease progression

 Applications

„ Molecular signatures of pathogens

„ Comparative genomic studies of pathogens

„ Virulence difference

„ Pathogen genetics and manifestation

„ Life cycle
„ Replication, translational control
 Applications

„ Host-parasite interaction

„ Pathogen establishment
„ Host cell recognition
„ Host cell response
„ Parasite response to host immune
response
Applications
 Constraints

„ Complex system of eukaryotes & multi-

cellular organisms

„ Transcriptome analysis

„ Developing technology

„ Many stages

„ Design of experiment
 Constraints

„ Array quality

„ Highly variable data

„ Analysis of data

„ Published experiments

„ Cost
 From Here to Tomorrow

„ Recent & Powerful

„ More improvement
„ Protocol
„ Hardware
„ Experimental design
„ Computational technique

„ Integrate with other data

„ Reproducible, fast, sensitive & economic