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1 DNA microarrays for gene expression profiling Alberto Bernacchia Dipartimento di Fisica E.Fermi, Università di Roma “La Sapienza”
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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
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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
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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)
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5 Example of scanned slide
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7 Alzheimer’s disease
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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
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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)
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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
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11 Example of scanned slide
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13 X
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14 1*backg 2* backg (50% of genes cut) 1*backg + 3 StDev (30% of genes cut)
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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)
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16 Clustering samples A distinction between two subtypes of cancer was discovered by clustering genes samples clustering of samples (columns)
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17 Clustering genes temporal response of human fibroblast to serum wound repair genes clustering of genes (rows)
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