Bioinformatics, 2012.11.15

Gene Expression Profiling by Microarray

Chun-Ju Chang, Ph.D.chunju@ntou.edu.twDepartment of Food ScienceCollege of Life SciencesNational Taiwan Ocean University

2Griffin & Shockcor . Nature Reviews Cancer 2004, 4:551.

High Throughput Gene Discovery• Solution for genomics study

3Sutliff J. Science 2001, 291:1224.

Gene chip (DNA chip, DNA microarray) Microarray technology evolved

from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known gene or fragment.

Nucleic Acids Res. 1992, 20:1679.

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Schematic of microarray analysisSchematic of microarray analysis

AnalysisAnalysisAnalysisAnalysis

Quality Quality measurementmeasurement Pre-ProcessingPre-Processing

FaileFailedd

PassedPassed

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Outline• Microarray platforms• Experimental design

– Sources of variability – Sample size and replication

• Data acquisition and preprocessing– Normalization– Quality control

• Data analysis– Partitional clustering– Functional annotation– Pathway analysis

• Gene expression databases

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Microarray platforms 1

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Microarray platforms 2

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Affymetrix

Miller & Tang. Microbiol Rev. 2009,22:611.

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Illumina

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250,000 probes/bead

Experimental design 1Sources of variation in a microarray experiment :• Manufacturing of arrays• Generation of biological sample

– Genetic and environmental factors– Pooled or individual samples – Randomization

• Technical variation – Preprocessing : RNA extraction, labeling, etc.– Protocolization of the processing steps

• Processing of samples – Obtaining image– “Biological replicates”, “technical replicates”

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Experimental design 2Sample size and replication:

• 4 types of experimental designs– Completely randomized treatment-control design: each

measurement is considered independent– Matched-pairs design– Multiple treatment design having an independent

treatment effect– Randomized block design

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Data acquisition and preprocessingCommon normalization strategies• Total intensity normalization• Normalization using regression techniques• Normalization using ratio statistics

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Data acquisition and preprocessingQuality control• From the Microarray Gene Expression Data (MGED) Society; presently

named Functional Genomics Data (FGED) Society• MIAME (Minimum Information About a Microarray Experiment)

standards for data reporting– Spotted cDNA and oligonucleotide arrays– Experimental design: number of replicates, samples used– Preparation and labeling– Hybridization procedures and parameters– Measurement data and specifications

• Microarray Gene Expression Markup Language (MAGE-ML)• ArrayExpress microarray database

– Universal data-presentation platform

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Functional Genomics Data (FGED) Society

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MicroArray Quality Control (MAQC) project

16Ji H & Davis RW. Nat Biotechnol 2006, 24:1112-3.

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Scatter plotHierarchical Trees

Pi Chart

K-MeansK-Means

Venn diagramVenn diagram

Partitional clustering by K-Means

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cluster centers, prototypes

反覆疊代

Functional annotation by GO (Gene Ontology)

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EMBL-EBI

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Hands-on Practice

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