1An Introduction to DNA microarraysRebecca Fry, Ph.D.(and Leona Samson)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 spots/genes=entire genome in 1 experimentA Revolution in Biology2mRNA Analysis Methodsz Northern Blot (Single Gene analysis) z Microarray Technology (Genome Wide Experiment)3•mRNAs separated on gel according to size•mRNAs transferred to a membrane and hybridized with small number (1-5) of radioactively labeled DNA probes. •Probe corresponds to gene of interest•Target RNA is spatially fixed and the labeled probe is in solution •Low throughputNORTHERN BLOTSi.e., your cloned gene(s)4Number of Genes in Different OrganismsYeast ~ 6200 genes E. coli ~ 4200 genesHuman ~ 30,000 genes Mouse ~ 30,000 genesPhage T4 ~ 200 genesInfluenza ~ 12 genes5i.e., your cloned gene(s)We could just 35,000 Northern to monitor expression of all genes!!!Northern BlotsImmobilized mRNA population hybridized with labeled probe representing one geneDNA MicroarraysImmobilized probes hybridized with labeled mRNA population representing all expressed genes6Need to achieve two things:(i)Immobilize thousands of probes specific for individual genes(ii)Label mRNA populationsEGFP ORF1846ATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGADesigning Oligo Probes70 mer oligospecific to gene of interest7Number of Genes in Different OrganismsYeast ~ 6200 genes E. coli ~ 4200 genesHuman ~ 30,000 genes Mouse ~ 30,000 genesPhage T4 ~ 200 genesInfluenza ~ 12 genesSpotted microarrayswww.biorobotics.comRobotic PrintingLiquid HandlingResuspension of oligoswww.qiageninstruments.comOverview of fabrication of spotted microarraysIntroduction8Need to achieve two things:(i)Immobilize thousands of probes specific for individual genes(ii)Label mRNA populations9562 nmcy3cy5www.amersham.comcy3cy5Differential dye incorporationcy5 less well than cy3Light sensitivity: cy5 more easily degradedProtect your reactions from light!!664 nm510 nmemissionemissionIntroductioncy3 and cy5: Commonly used dyes10www.genetics.ucla.eduLabeled cDNA preparationRNASample 2Spotted MicroarrayTarget preparationReverse transcriptionFlourescent dyesIntroductionEGFP KDCombined in equal amountsSpotted microarray target preparationDirect labelingcy5cy3cDNAcDNAEGFPCo-hybridized to arraywww.genetics.ucla.eduLabeled cDNA preparationRNASample 2Spotted MicroarrayTarget preparationReverse transcriptionFlourescent dyesIntroductionCombined in equal amountsSpotted microarray target preparationDirect labelingcy5cy3cDNAcDNACo-hybridized to arrayyellow cy3=cy5red cy5>cy3green cy3>cy5EGFP KDEGFP11cDNA “Two Color Chips”Target preparation Array preparationsummaryThis is the kind of thing you will see in YOUR microarray experiments12What’s happening at each spot?cDNA Chip vs. Northern Blot13Two Popular Microarraying Platformswww.molgen.mpg.deSpotted microarrayscDNA: PCR products (500-1,000bp)synthesized oligos (70 mer)>10,000 probesAffymetrix“Gene Chip”500,000 probes25 mer(represents a fragment of a gene)IntroductionCommercially available Oligo microarraywww.the-scientist.comSo..what gene probes are represented on the array you will use??EGFP, p53, EXO1, AAG, ATM, and ATR….But we added in a bunch more!!14BRCA2BRCA1BLMBIDATRATMAPEXL2APEXADPRTL3ADPRTL2ADPRTABH3ABH2ABHDDB1DCLRE1ACETN2CDK8CDK7CDK6CDK4CDK2CCNHCASP9CASP8CASP3CASP2BTG1EXO1FANCAFANCCFANCEFANCFERCC5ERCC4ERCC3ERCC2ERCC1ENDOGEGFPDUTDMC1MAD2L2LIG4LIG3LIG1JUNHUS1HSU24186HCNPHAP1GTF2H4GADD45GGADD45BGADD45AG22P1POLQPRKDCPRSS25RAD1RAD17RAD18RAD23ARAD23BRAD50RAD51RAD51CRAD51L1RAD51L3RAD52RAD54BalkB homologBreast cancer 1Excision repairGrowth arrest and DNA-damage-inducibleNo homology toHuman sequencesAlien DNA1528SLiver 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, 1,2Gel 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