Gene Expression Gene Expression DataData

Qifang XuQifang Xu

OutlineOutline

cDNA Microarray TechnologycDNA Microarray Technology Data RepresentationData Representation Statistical Analysis of cDNA Statistical Analysis of cDNA

Microarray DataMicroarray Data Oligonucleotide Microarray Oligonucleotide Microarray

TechnologyTechnology Microarray ApplicationsMicroarray Applications Microarray DatabasesMicroarray Databases

cDNA MicroarraycDNA Microarray

Measure the relative levels of Measure the relative levels of expression expression

Parallel analysisParallel analysis Competitive hybridizationCompetitive hybridization Need cDNA libraryNeed cDNA library

mRNA cDNAmRNA cDNA

Reverse Transcription

PCR Amplification

Printing

Hybridization

Laser Scan

Labeling

SamplesReverse Transcription

Expression Data

Exponential Amplification of a Exponential Amplification of a GeneGene

Return

Labeling and Hybridization Labeling and Hybridization

of Sample cDNAsof Sample cDNAs

Return

Data DescriptionData Description Ratios, why?Ratios, why?

Competitive hybridization Competitive hybridization no absolute expression levels no absolute expression levels

Gene expression levels determined by intrinsic properties of each gene

low high expression level

Gene A Gene B

Sample 2

Sample 1

Data NormalizationData Normalization

PurposePurposeAdjust bias from variation in microarray Adjust bias from variation in microarray technology.technology.

E.g. differences between labeling, scanner setting, E.g. differences between labeling, scanner setting, spatial positionsspatial positions

Within-array normalizationWithin-array normalizationlogarithmic transformation of ratio, subtract by logarithmic transformation of ratio, subtract by mean log ratiomean log ratio

Red Green Difference Ratio (G/R) Log2 Ratio Centered R

16500 15104 -1396 0.915 -0.128 -0.048

357 158 -199 0.443 -1.175 -1.095

8250 8025 -225 0.973 -0.039 0.040

978 836 -142 0.855 -0.226 -0.146

65 89 24 1.369 0.453 0.533

684 1368 529 2.000 1.000 1.080

13772 11209 -2563 0.814 -0.297 -0.217

856 731 -125 0.854 -0.228 -0.148

Statistical AnalysisStatistical Analysis

• Differences in ratios due to– random variation

– meaningful changes

• Convention

– ratio >= 2 or ratio <= ½

• Analysis of variance (ANOVA)– 4 and 10 replicates of each treatment

– statistical significance

Oligonucleotide Oligonucleotide MicroarrayMicroarray

Parallel analysis of gene expressionParallel analysis of gene expression Unit of hybridization: oligonucleotidesUnit of hybridization: oligonucleotides

represent known or predicted open reading frames (ORF)represent known or predicted open reading frames (ORF)

Noncompetitive hybridizationNoncompetitive hybridization Data RepresentationData Representation

average differenceaverage difference between perfect match and mismatch between perfect match and mismatch intensitiesintensities

Difference in expression levels between Difference in expression levels between two samplestwo samplescomparison across chipscomparison across chips

Oligonucleotide Oligonucleotide MicroarrayMicroarray

Construction of Construction of oligonucleotide arraysoligonucleotide arrays

Comparison between cDNA and Comparison between cDNA and Oligonucleotide MicroarrayOligonucleotide Microarray

cDNA MicroarraycDNA Microarray cheaper cheaper replicates replicates hybridization over kilobases (1 – 2 kb)hybridization over kilobases (1 – 2 kb)

reduce cross-hybridizationreduce cross-hybridization genome sequence unknowngenome sequence unknown cDNA librarycDNA library Competitive hybridizationCompetitive hybridization

Oligonucleotide MicroarrayOligonucleotide Microarray accommodate higher densities of genes accommodate higher densities of genes

predicted genes not in cDNA predicted genes not in cDNA librarylibrary

lower variabilitylower variability incorporate mismatch controls incorporate mismatch controls

reduce possibility of cross-reduce possibility of cross-hybridizationhybridization

Sequence knownSequence known Noncompetitive hybridizationNoncompetitive hybridization

Microarray ApplicationsMicroarray Applications

Drug Discovery – Identification of drug targets to treat diseases

Drug Development – rule out potential drug candidates before entering trials

Diagnostics and Disease Treatment – Screening and identification of patient samples

Model Organismsmouse brain modelLife cycle of a fruitfly

Genetic Regulatory NetworkGenetic Regulatory Network

Gene Expression Gene Expression DatabasesDatabases

Stanford Microarray Database (SMD)Stanford Microarray Database (SMD) http://genome-www5.stanford.edu/MicroArray/SMhttp://genome-www5.stanford.edu/MicroArray/SM

D/D/ raw and normalized data, image filesraw and normalized data, image files

The Gene Expression Database (GXD)The Gene Expression Database (GXD) http://http://

www.informatics.jax.org/mgihome/GXD/aboutGXD.www.informatics.jax.org/mgihome/GXD/aboutGXD.shtmlshtml

Endogenous gene expression during mouse Endogenous gene expression during mouse development.development.

ExpressDB ExpressDB http://http://twod.med.harvard.edu/ExpressDBtwod.med.harvard.edu/ExpressDB// Relational database for yeast and E. coli RNA Relational database for yeast and E. coli RNA

expression dataexpression data

Thank you !Thank you !