1. 1 DNA microarrays for gene expression profiling Alberto Bernacchia Dipartimento di Fisica E.Fermi, Università di Roma “La Sapienza”

2. 2 Motivations We know entire DNA sequences (genomes) of different organisms. How are the genes expressed in different conditions? DNA microarrays may answer this question

3. 3 DNA microarray manufacture On each slide (few centimeters large), ≈10 5 spots are deposited (≈1  m). On each spot, a single gene is chemically bounded to the surface. The entire genome in one slide few hundred dollars each

4. 4 An experiment Reference sample Test sample RNA extraction cDNA synthesis (RT) and amplification cRNA synthesis and labeling Cy5 Cy3 Hybridization DNA / RNA Laser Scanning (healthy) (diseased)

5. 5 Example of scanned slide

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7. 7 Alzheimer’s disease

8. 8 European Brain Research Institute (EBRI) Istituto di Analisi dei Sistemi ed Informatica (IASI) AD11 transgenic mices exepress the Alzheimer’s cellular phenotype Alzheimer’s desease

9. 9 Three brain areas: cerebral cortex, hippocampus, basal forebrain. 15 months 6 months 3 months 1 month Experimental setting Four ages Total 4x3x2=24 conditions Two mices: anti-NGF AD11 (diseased), VH  D11 (healthy)

10. 10 Reproducibility of arrays Two conditions healthy diseased More conditions diseased healthy reference (wild) healthy, hyppocampus, 1 month reference (wild) diseased, cerebral cortex, 3 months reference (wild) healthy, basal forebrain, 6 month reference (wild) diseased, hyppocampus, 15 months 24 conditions x 5 = 120 microarrays

11. 11 Example of scanned slide

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13. 13 X

14. 14 1*backg 2* backg (50% of genes cut) 1*backg + 3 StDev (30% of genes cut)

15. 15 Log 2 (Red/Green) Intensity =Red x Green Lowess (Locally Weighted Linear Regression): data shift based on non-linear fitting Lowess normalization Intensity =Red x Green Log 2 (Red/Green)

16. 16 Clustering samples A distinction between two subtypes of cancer was discovered by clustering genes samples clustering of samples (columns)

17. 17 Clustering genes temporal response of human fibroblast to serum wound repair genes clustering of genes (rows)