1. Gene Expression Data Qifang Xu

2. Outline cDNA Microarray Technology cDNA Microarray Technology Data Representation Data Representation Statistical Analysis of cDNA Microarray Data Statistical Analysis of cDNA Microarray Data Oligonucleotide Microarray Technology Oligonucleotide Microarray Technology Microarray Applications Microarray Applications Microarray Databases Microarray Databases

3. cDNA Microarray Measure the relative levels of expression Measure the relative levels of expression Parallel analysis Parallel analysis Competitive hybridization Competitive hybridization Need cDNA library Need cDNA library mRNA cDNA Reverse Transcription

4. PCR Amplification Printing Hybridization Laser Scan Labeling Samples Reverse Transcription Expression Data

6. Exponential Amplification of a Gene Return

7. Labeling and Hybridization of Sample cDNAs Return

8. Data Description Ratios, why? Ratios, why? Competitive hybridization  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

9. Data Normalization Purpose Purpose Adjust bias from variation in microarray technology. E.g. differences between labeling, scanner setting, spatial positions E.g. differences between labeling, scanner setting, spatial positions Within-array normalization Within-array normalization logarithmic transformation of ratio, subtract by mean log ratio

10. Statistical 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

11. Oligonucleotide Microarray Parallel analysis of gene expression Parallel analysis of gene expression Unit of hybridization: oligonucleotides Unit of hybridization: oligonucleotides represent known or predicted open reading frames (ORF) Noncompetitive hybridization Noncompetitive hybridization Data Representation Data Representation average difference between perfect match and mismatch intensities Difference in expression levels between two samples Difference in expression levels between two samples comparison across chips

12. Oligonucleotide Microarray

13. Construction of oligonucleotide arrays

14. Comparison between cDNA and Oligonucleotide Microarray cDNA Microarray cDNA Microarray cheaper  replicates cheaper  replicates hybridization over kilobases (1 – 2 kb) hybridization over kilobases (1 – 2 kb)  reduce cross-hybridization genome sequence unknown genome sequence unknown cDNA library cDNA library Competitive hybridization Competitive hybridization Oligonucleotide Microarray Oligonucleotide Microarray accommodate higher densities of genes accommodate higher densities of genes  predicted genes not in cDNA library lower variability lower variability incorporate mismatch controls incorporate mismatch controls  reduce possibility of cross-hybridization Sequence known Sequence known Noncompetitive hybridization Noncompetitive hybridization

15. Microarray 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 Organisms mouse brain model Life cycle of a fruitfly Genetic Regulatory Network Genetic Regulatory Network

16. Gene Expression Databases Stanford Microarray Database (SMD) Stanford Microarray Database (SMD) http://genome-www5.stanford.edu/MicroArray/SMD/ http://genome-www5.stanford.edu/MicroArray/SMD/ http://genome-www5.stanford.edu/MicroArray/SMD/ raw and normalized data, image files raw and normalized data, image files The Gene Expression Database (GXD) The Gene Expression Database (GXD) http://www.informatics.jax.org/mgihome/GXD/aboutGXD.sht ml http://www.informatics.jax.org/mgihome/GXD/aboutGXD.sht ml http://www.informatics.jax.org/mgihome/GXD/aboutGXD.sht ml http://www.informatics.jax.org/mgihome/GXD/aboutGXD.sht ml Endogenous gene expression during mouse development. Endogenous gene expression during mouse development. ExpressDB ExpressDB http://twod.med.harvard.edu/ExpressDB/ http://twod.med.harvard.edu/ExpressDB/ http://twod.med.harvard.edu/ExpressDB/ Relational database for yeast and E. coli RNA expression data Relational database for yeast and E. coli RNA expression data

17. Thank you !