Gene Expression BMI 731 week 5Thesis: the analysis of gene expression data is going to be big in 21st century statisticsSlide 3Common themesBiological backgroundSlide 6Reverse transcriptioncDNA microarray experimentsSlide 9Affymetrix® Instrument SystemPhotolithographySynthesis of Ordered Oligonucleotide ArraysAffymetrix GeneChip arraysGeneChip® Probe ArraysSlide 15Analysis of expression level from probe setsExpression screening by GeneChipAffymetrix GeneChip arrayscDNA microarrayscDNA microarrayshttp://www.bio.davidson.edu/courses/genomics/chip/chip.swfSlide 22Some statistical questionsSome statistical questions, ctdSome bioinformatic questionsSlide 26Slide 27Segmentation: limitation of the fixed circle methodSome local backgroundsQuantification of expressionGene Expression DataSlide 32The red/green ratios can be spatially biasedAffymetrix vs. cDNA ArraysAffymetrix weaknesses/limitationsLimitations to all microarrays.AnalysisNormalizationSlide 39The simplest cDNA microarray data analysis problem is identifying differentially expressed genes using replicated slidesApo AI experiment (Matt Callow, LBNL)Slide 42Which genes have changed? When permutation testing possibleHistogram & normal q-q plot of t-statisticsPatterns, More Globally...Slide 46Slide 47Slide 48Gene ExpressionBMI 731 week 5 Catalin BarbacioruDepartment of Biomedical InformaticsOhio State UniversityThesis: the analysis of gene expression data is going to be big in 21st century statisticsMany different technologies, includingHigh-density nylon membrane arraysSerial analysis of gene expression (SAGE)Short oligonucleotide arrays (Affymetrix) Long oligo arrays (Agilent)Fibre optic arrays (Illumina)cDNA arrays (Brown/Botstein)*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…..databasesBiological backgroundG T A A T C C T C | | | | | | | | | C A T T A G G A GDNAG U A A U C CRNA polymerasemRNATranscriptionIdea: 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.Reverse transcriptionClone cDNA strands, complementary to the mRNAG U A A U C C U CReverse transcriptasemRNAcDNA C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A GT T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A GcDNA microarray experiments mRNA levels compared in many different contextsDifferent tissues, same organism (brain v. liver) Same tissue, same organism (ttt v. ctl, tumor v. non-tumor) Same tissue, different organisms (wt v. ko, tg, or mutant)Time course experiments (effect of ttt, development)Other special designs (e.g. to detect spatial patterns).•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 CellA single, contiguous gene set for the rat B-actin gene.Perfect 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 setsExpression screening by GeneChip• 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 synthesizedonto a 2 x 2 cm glass substrateAffymetrix GeneChip 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.cDNA 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