Parallel human genome analysis: Microarray-based expression monitoring of 1000 genes Mark Schena,...
-
date post
20-Dec-2015 -
Category
Documents
-
view
213 -
download
0
Transcript of 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
Schena et. alSchena et. al
Types of MicroarraysTypes of Microarrays Photolithography – using oligosPhotolithography – using oligos Spotting – using cDNAs/ESTsSpotting – using cDNAs/ESTs
Schena et. alSchena et. al
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
Schena et. al, Figure 2Schena et. al, Figure 2
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
Schena et. al, Figure 3Schena et. al, Figure 3
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
Schena et. alSchena et. al
Schena et. alSchena et. al
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
Schena et. alSchena et. al