parallel human genome analysis: microarray-based expression monitoring of 1000 genes mark schena,...

Parallel human genome Parallel human genome analysis: Microarray-analysis: Microarray-

based expression based expression monitoring of 1000 genesmonitoring of 1000 genes

Mark Schena, Dari Shalon, Renu Mark Schena, Dari Shalon, Renu Heller, Andrew Chai, Patrick O. Heller, Andrew Chai, Patrick O.

Brown, and Ronald W. DavisBrown, and Ronald W. Davis

GoalsGoals To detect the expression of thousands of To detect the expression of thousands of

genes simultaneouslygenes simultaneously Gene expression studies – expression Gene expression studies – expression

patterns of genes provide clues to function by patterns of genes provide clues to function by comparisoncomparison

Gene discovery studiesGene discovery studies

Use the microarray assay to identifyUse the microarray assay to identify Known and novel heat shock genesKnown and novel heat shock genes Phorbol-ester regulated genesPhorbol-ester regulated genes

Schena et. alSchena et. al


Types of MicroarraysTypes of Microarrays Photolithography – using oligosPhotolithography – using oligos Spotting – using cDNAs/ESTsSpotting – using cDNAs/ESTs


Human cDNA from human T mRNA Human cDNA from human T mRNA transformed by the Epstein Barr Virus transformed by the Epstein Barr Virus with 5’ amino acid modification, amplified with 5’ amino acid modification, amplified by PCR, and arrayed onto silyated by PCR, and arrayed onto silyated microscope slidesmicroscope slides Probes labeled with fluorescin and Cy5-dCTP Probes labeled with fluorescin and Cy5-dCTP

are hybridized to 1056-element array and are hybridized to 1056-element array and scannedscanned

Verify expression patterns with RNA BlotVerify expression patterns with RNA Blot Array elements that display differential Array elements that display differential

expression patterns are sequenced expression patterns are sequenced Compare sequence to Informatics databasesCompare sequence to Informatics databases

MethodsMethods

Schena et. al, Figure 1Schena et. al, Figure 1

Monitoring of heat shock response in Monitoring of heat shock response in control (37control (37oo)and treated Jurkat (43)and treated Jurkat (43oo,,

human T) cells human T) cells - Array contains 10 Arabidopsis controls, 1046 human blood cDNAs-White box indicates altered fluorescence:

-Red boxes indicate activation

-Green boxes indicate repression

Hybridization signals observed for > 95% of human Hybridization signals observed for > 95% of human cDNA elementscDNA elementsComparative expression finds altered fluorescence in Comparative expression finds altered fluorescence in 17 array elements17 array elements


Elemental displays of activated and Elemental displays of activated and repressed genesrepressed genes Fluorescin labeled

probes from (+) heat-shock and (+) phobol ester cells are compared to Cy5-labeled untreated probes

Data is the average of the ratios from the 2 hybridizations

17 elements have a 2-fold alteration in fluorescence

Intensity of fluorescnece is a measure of mRNA abundance

Schena et. al, Table 1Schena et. al, Table 1

14/17 clones matched; proximal and 14/17 clones matched; proximal and distal ends map to same genedistal ends map to same geneHsp90, dnaJ, polyubiquitin, tcp-1 are Hsp90, dnaJ, polyubiquitin, tcp-1 are highly inducedhighly inducedNovel sequences (B7-B9) have 2-fold Novel sequences (B7-B9) have 2-fold inductioninduction

Schena et. al, Table 2Schena et. al, Table 2

Correlation of human gene expression from Correlation of human gene expression from microarray analysis is confirmed by RNA blot microarray analysis is confirmed by RNA blot

analysesanalyses


Microarrays measureMicroarrays measureexpression in human tissuesexpression in human tissues

Bone marrow, brain, prostate, and heartBone marrow, brain, prostate, and heart

Expression levels in genes correlates with Expression levels in genes correlates with expression level in tissuesexpression level in tissues

AdvantagesAdvantages Small hybridization volumes using Small hybridization volumes using

cDNA provides specificity not possible cDNA provides specificity not possible with oligo-based arrayswith oligo-based arrays

High array densitiesHigh array densities Incorporation of fluorescence labeling Incorporation of fluorescence labeling

and detectionand detection High throughput: sequence-based High throughput: sequence-based

methods require serial processingmethods require serial processing Rich number of ESTs makes for more Rich number of ESTs makes for more

powerful arrayspowerful arrays



DisadvantagesDisadvantages CostCost Commercial availability of microarraysCommercial availability of microarrays

ConclusionsConclusions Microarrays are useful for gene discovery in Microarrays are useful for gene discovery in

the absence of sequence informationthe absence of sequence information Parallel assays can monitor gene expression for Parallel assays can monitor gene expression for

thousands of genesthousands of genes Allows high throughput human genome expression Allows high throughput human genome expression

and gene discoveryand gene discovery Allows for rapid mechanistic examination of Allows for rapid mechanistic examination of

hormones, drugs, elicitors, and other small hormones, drugs, elicitors, and other small moleculesmolecules

Potential capacities for patient screeningPotential capacities for patient screening Sensitivities of microarrays allows for Sensitivities of microarrays allows for

functional analysis of transcription factors, functional analysis of transcription factors, kinases, growth factorskinases, growth factors


parallel human genome analysis: microarray-based expression monitoring of 1000 genes mark schena,...

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