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OSU BMI 731 - Gene Expression

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Gene Expression BMI 731 Winter 2005Thesis: the analysis of gene expression data is going to be big in 21st century statisticsSlide 3Common themesCentral dogmaSlide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Affymetrix® Instrument SystemPhotolithographySynthesis of Ordered Oligonucleotide ArraysAffymetrix GeneChip arraysGeneChip® Probe ArraysSlide 23Slide 24Analysis of expression level from probe setsSlide 26Affymetrix arraysSlide 28Slide 29Spotted DNA microarraysSpotted DNA microarrays 33Slide 34Slide 35Slide 36Slide 37The red/green ratios can be spatially biasedSlide 39Spotted vs. Affymetrix ArraysAffymetrix weaknesses/limitationsLimitations to all microarraysSlide 43Slide 44Slide 45Slide 46Slide 47Slide 48Gene ExpressionBMI 731 Winter 2005 Catalin BarbacioruDepartment of Biomedical InformaticsOhio State UniversityThesis: the analysis of gene expression data is going to be big in 21st century statisticsMany different technologies, includingSpotted DNA arrays (Brown/Botstein)Short oligonucleotide arrays (Affymetrix) Serial analysis of gene expression (SAGE)Long oligo arrays (Agilent)Fibre optic arrays (Illumina)1995 1996 1997 1998 1999 2000 20010100200300400500600(projected)YearNumber of papersTotal microarray articles indexed in MedlineCommon themes•Parallel approach to collection of very large amounts of data (by biological standards)•Sophisticated instrumentation, requires some understanding•Systematic features of the data are at least as important as the random ones•Often more like industrial process than single investigator lab research•Integration of many data types: clinical, genetic, molecular…..databasesCentral dogmaThe expression of the genetic information stored in the DNA molecule occurs in two stages:•(i) transcription, during which DNA is transcribed into mRNA; •(ii) translation, during which mRNA is translated to produce a protein.DNA → mRNA → protein Other important aspects of gene regulation: methylation, alternative splicing.Idea: measure the amount of mRNA to see which genes are being expressed in (used by) the cell.Measuring protein might be better, but is currently harder.•DNA microarrays represent an important new method for determining the complete expression profile of a cell. •Monitoring gene expression lies at the heart of a wide variety of medical and biological research projects, including classifying diseases, understanding basic biological processes, and identifying new drug targets.Affymetrix® Instrument System Platform for GeneChipPlatform for GeneChip®® Probe Arrays Probe Arrays •IntegratedIntegrated•Easy to useEasy to use•ExportableExportable•VersatileVersatilePhotolithographySynthesis of Ordered Oligonucleotide ArraysO O O O OLight(deprotection)HO HO O O OT T O O OT T C C OLight(deprotection)T T O O OC A T A TA G C T GT T C C GMaskMaskSubstrateSubstrateMaskMaskSubstrateSubstrateT –T –C –C –REPEATREPEATAffymetrix GeneChip arraysGeneChip® Probe Arrays24µm24µmMillions of copies of a specificMillions of copies of a specificoligonucleotide probeoligonucleotide probe Image of Hybridized Probe ArrayImage of Hybridized Probe Array>200,000 different>200,000 differentcomplementary probes complementary probes Single stranded, Single stranded, labeled RNA targetlabeled RNA targetOligonucleotide probeOligonucleotide probe*****1.28cm1.28cmGeneChipGeneChip Probe ArrayProbe ArrayHybridized Probe CellHybridized Probe CellPerfect Match (PM)Mis Match (MM) Controllog(PM / MM) = difference scoreAll significant difference scores are averaged to create “average difference” = expression level of the gene.Each pixel is quantitated and integrated for each oligo feature (range 0-25,000)Analysis of expression level from probe sets• each oligo sequence (20-25 mer) is synthesized as a 20 µ square (feature)• each feature contains > 1 million copies of the oligo• scanner resolution is about 2 µ (pixel)• each gene is quantitated by 16-20 oligos andcompared to equal # of mismatched controls• 22,000 genes are evaluated with 20 matching oligosand 10 mismatched oligos = 480,000 features/chip• 480,000 features are photolithographically synthesized onto a 2 x 2 cm glass substrateAnalysis of expression level from probe setsAffymetrix arrays•Global views of gene expression are often essential for obtaining comprehensive pictures of cell function. •For example, it is estimated that between 0.2 to 10% of the 10,000 to 20,000 mRNA species in a typical mammalian cell are differentially expressed between cancer and normal tissues. •Whole-genome analyses also benefit studies where the end goal is to focus on small numbers of genes, by providing an efficient tool to sort through the activities of thousands of genes, and to recognize the key players. •In addition, monitoring multiple genes in parallel allows the identification of robust classifiers, called "signatures", of disease. •Global analyses frequently provide insights into multiple facets of a project. A study designed to identify new disease classes, for example, may also reveal clues about the basic biology of disorders, and may suggest novel drug targets.Spotted DNA microarrays •In ‘‘spotted’’ microarrays, slides carrying spots of target DNA are hybridized to fluorescently labeled cDNA from experimental and control cells and the arrays are imaged at two or more wavelengths •Expression profiling involves the hybridization of fluorescently labeled cDNA, prepared from cellular mRNA, to microarrays carrying thousands of unique sequences. •Typically, a set of target DNA samples representing different genes is prepared by PCR and transferred to a coated slide to form a 2-D array of spots with a center-to-center distance (pitch) of about 200 μm, providing a pan-genomic profile in an area of 3 cm2 or less.•cDNA samples from experimental and control cells are labeled with different color fluors (cytochrome Cy5 and Cy3) and hybridized simultaneously to microarrays, and the relative levels of mRNA for each gene are then determined by comparing red and green signal intensitiesSpotted DNA microarraysScanning Technology•Microarray slides are imaged with a modified fluorescence microscope designed for scanning large areas at high resolution (arrayWoRx, Applied Precision, Issaquah, WA,

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