1An Introduction to DNA microarraysRebecca Fry, Ph.D.http://www.buffalo.edu/UBT/UBTWhat is a DNA Microarray?genes or gene fragments attached to a substrate (glass)Hybridized slideTwo dyesImage analyzed Tens of thousands of spots2The Beginnings of Microarray TechnologyLockhart et al., 1996Nature Biotechnology“Expression monitoring by hybridisation to high-density oligonucleotidearrays”Schena et al., 1995 Science“Quantitative monitoring of gene expression patterns with a complementary DNA microarray”8 years later4162 references3Uses and ApplicationsDisease characterizationGene screeningPathway mappingTarget identificationDevelopmental BiologyPersonalized medicineMechanism of Action Studies Molecular Diagnosis of DiseaseToxicologyPrediction of Drug Efficacy/ToxicityA model experiment: Two samples of interestQ: Which genes are differentially expressed in EGFP cells versus EGFP KD cells?EGFP KDEGFP expressing cellshttp://www.oardc.ohio-state.edu/plantranslab/gfp-a.gifDifferential Gene Expression?!4CellnucleuscytoplasmDNAInside every cell: DNA Serves as a genetic blueprintRNAProteinChem.ox.ac.ukDna molecule: berkeley.eduProcess of transcribing deoxyribonucleic acid toribonucleic acid is“TRANSCRIPTION”Nucleic acidsmust be translated intoamino acids that make up proteinsProcess: “TRANSLATION”RNAProteinDNARelating Gene ExpressionIs a gene expressed? Is protein producedIdeally measure protein levelsHigh throughput protein assays complicatedWe measure transcript level5Two Popular Microarraying Platformswww.molgen.mpg.deSpotted microarrayscDNA: PCR products (500-2,000bp)synthesized oligos>10,000 probesAffymetrix“Gene Chip”500,000 probes25 mer (represents a fragment of a gene)IntroductionCommercially available microarraywww.the-scientist.comEGFP ORF1846ATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGADesigning Oligos70 mer oligospecific to gene of interest6Spotted microarrayswww.biorobotics.comRobotic PrintingRobotics: Liquid HandlingResuspension of oligoswww.qiageninstruments.comOverview of fabrication of spotted microarraysIntroductionBRCA2BRCA1BLMBIDATRATMAPEXL2APEXADPRTL3ADPRTL2ADPRTABH3ABH2ABHDDB1DCLRE1ACETN2CDK8CDK7CDK6CDK4CDK2CCNHCASP9CASP8CASP3CASP2BTG1EXO1FANCAFANCCFANCEFANCFERCC5ERCC4ERCC3ERCC2ERCC1ENDOGEGFPDUTDMC1MAD2L2LIG4LIG3LIG1JUNHUS1HSU24186HCNPHAP1GTF2H4GADD45GGADD45BGADD45AG22P1POLQPRKDCPRSS25RAD1RAD17RAD18RAD23ARAD23BRAD50RAD51RAD51CRAD51L1RAD51L3RAD52RAD54BalkB homologBreast cancer 1Excision repairGrowth arrest and DNA-damage-inducible7A closer look at Spotted microarraysSome nomenclaturewww.molgen.mpg.deeach spotrepresents a gene or gene fragmentgeneRNA“probe”“target”Introduction562 nmcy3cy5www.amersham.comcy3cy5Differential dye incorporationcy5 less well than cy3Light sensitivity: cy5 more easily degraded664 nm510 nmemissionemissionIntroductioncy3 and cy5: Commonly used dyes8www.genetics.ucla.eduLabeled cDNA preparationRNASample 2Spotted MicroarrayTarget preparationReverse transcriptionFlourescent dyesIntroductionEGFP KDCombined in equal amountsSpotted microarray target preparationDirect labelingcy5cy3cDNAcDNAEGFPCo-hybridized to arrayyellow cy3=cy5red cy5>cy3green cy3>cy5DNADirect Labeling (Spotted Arrays)RNARTReverse transcriptionRNAEnzyme (Superscript RT)DyeOligo d(T)nucleotidesAAAAAAAAAAAAAAAAAAEGFP KDEGFP928SLiver 18S28S6 kb4 kb2 kb1 kb0.5 kb0.2 kbElectropherogram (28S/18S Ratio~2)18S28SFluorescenceTime (seconds)010203040506070 19 24 29 34 39 44 49 54 59 64 69Sample 1 Ladder Sample 2RNA quality controlLad, L, PGel Image (in silico) Sharp, Clear BandsPre-labeling quality control:Determine RNA Quality Agilent Bioanalyzer: 50-500 ngNo more formaldehyde gels!! Microarray MeasurementsScannerImage Analysis.txt or .xls fileImage Analysis: Spotted arrays10Spotted microarrayswww.molgen.mpg.deSignal is average of pixel intensities of spot2 numbers per spotMicroarray Measurementscy5cy3Signal: Spotted arraysInformation: Gene nameOligo idCy3 intensityCy5 intensityImage Analysis: Spotted arraysWhat information do we see?117.8919272.5676H200008289ADPRT108.9855259.5362H200008289ADPRT108.5797265.7391H200008289ADPRT131.2286324.886H200008289ADPRT115.6119296.5224H200014101PRKDC117.8676306.8235H200014101PRKDC129.5333340.933H200014101PRKDC132348.522H200014101PRKDC2351.6976242.5control1control12370.5756434.93control1control12068.515786.72control1control12371.1636642.5control1control1Spot Mean Intensity (w685)Spot Mean Intensity (w595)Clone IDNameSteps:Normalize globally Calculate average of cy3 and cy5Bring cy5 numbers to cy3 by multiplying by common factorRatios (take ratio of wt/KD…look for 2 fold cutoff)Log2 (calculate log 2 of ratio to differentiate increase or decrease)Reproducibility (how did the four replicates perform?)11Scatter PlotdChip expressiontiss diff_ID0 2000 4000 6000 8000 10000 12000-9-8-7-6-5-4-3-2-101234567892 fold increase2 fold decreaseGene populationPlotsEGFP versus EGFP KD Ratio Log2 Data AnalysisRequires software: SpotfireRequires ability to search for Patterns and Trends12mammalian DNA damage response pathwayThe DNA damage response: puttingcheckpoints in perspectiveBin-Bing S. Zhou* & Stephen J. Elledge²NATURE |VOL 408 | 23 NOVEMBER 2000 |Good