ToxicogenomicsToxicogenomicsGene Expression Studies:• Pattern of genes expressed in a cell is characteristic of its current state • Many differences in cell state or type are correlated with changes in mRNA levels of many genes • Expression patterns of many previously uncharacterized genes may provide clues to their possible function by comparison with how known genes act• Gene expression data can be combined with metabolic schemas to understand how pathways are changed under varying conditions (i.e., mechanisms of action)Biology & Functional Genomics:• Having information on the level of expression of all genes that are present in a genome is very good• But genetics is the study of the interactions of these individual genes in an organism • Want to study complex interplay of all genes simultaneously• This requires high-throughput and large-scale technologies to study gene expression of all genes simultaneouslyToxicogenomics is a new scientific field that elucidates how the entire genome is involved in biological responses of organisms exposed to environmental toxicants/stressors http://www.niehs.nih.gov/multimedia/qt/ntc/ntcaltcaption.movhttp://video.niehs.nih.gov:7075/ramgen/ntc/ntc-eng03.rmhttp://www.niehs.nih.gov/nct/home.htmDNA Microarray TechnologyDNA Microarray TechnologyHas many potential applications: ► Studying changes in expression of genes over time, between tissues, and disease states ► Identification of complex genetic diseases ► Drug discovery and toxicology studies ► Mutation/polymorphism detection (SNP’s) ► Pathogen analysisDNA complementary to genes of interest is generated and laid out in microscopic quantities on solid surfaces at defined positionsPresence of bound DNA is detected by fluorescence following laser excitationcDNAs (from mRNA) from samples are eluted over the surface Æ complementary DNA bindsMicroarray Experiments:Microarray Experiments:analysis of gene expressionanalysis of gene expression• Analyze cell signaling networks (e.g., cell-cycle genes)• Determine effects of various exposures/conditions• Predict/discover function of unknown genes• Compare “normal” to “abnormal” (e.g., tumor cells): Analyze expression patterns  Novel gene association/discovery Divide tumors into sub-classes Determine effects of treatment• Genome-wide (e.g., yeast)• Partial selection of known/unknown genesScientific Areas in ToxicogenomicsScientific Areas in ToxicogenomicsDisease Mechanisms: application of gene expression profiling technologies to define the mechanistic underpinnings of environmentally related diseases; genetic and environmental components of disease, elucidation of disease pathways and networks, and development of disease models. Susceptibility: individual and population susceptibilities to exposure and disease as derived from genetic and environmental analysis and integration; identification of gene targets and factors mediating susceptibility, and gender-, strain- and species-susceptibility.Comparative Genomics: comparative and integrated responses of organisms to environmental stimuli; cross-species comparisons of biological responses to environmental factors at the gene, transcription, and protein level and their integration in model organisms; conserved biological components, pathways and responses to environmental factors; and computational tools to support comparative toxicogenomics.Predictive Toxicology: development and application of gene expression, proteomics and metabolomics technologies in predictive toxicology; development of model systems and research tools, and linkage of predictive responses to disease phenotype.Classical Microarray ExperimentsClassical Microarray Experiments•Normal vs DiseaseExample: Analysis of expression patterns in cancer-Pattern of gene expression-networks-Novel gene association/discovery•Molecular ClassificationExample: Comparison of Breast Tumors-Samples classified into subtypes •Genome-Wide AnalysisExample: Genome-wide expression in S. cerevisiaeMicroarray TechniqueMicroarray TechniqueMicroarray: A substrate with bound capture probesCapture probe: An oligo/cDNA with gene (DNA sequence) of interestGeneric experimental steps:1. Fabrication:Photolithography Affymetrix (one-color array) >40,000 genesPrinting Agilent (two-color arrays) ~45,000 genesSpotting In-house (two-color arrays) <24,000 genes2. Target Generation from a sample of interest:One color (biotin labeled cRNA, phycoerythrin-streptavidin detection) Two color (Cy3 and Cy5 cDNA labeling)3. Hybridization4. Analysis“Scanning” of arrayAmount of hybridized target is assessedStatistical interrogation of the dataSpotted Array ConstructionFabrication of twoFabrication of two--color color oligooligo/cDNA arrays/cDNA arraysOligos (20-70 bp)or cDNAs96-well plate 384-well plate printed on a glass slide150 µm200 µmTwoTwo--color color oligooligo/cDNA arrays/cDNA arraysTwo mRNA sources to be compared are labeled with fluorescent probes:Cy3 (green) used for one sample (e.g., “control”)Cy5 (red) used for the other (e.g., “treatment”)Probes are mixed and washed over the microarray (hybridization)Each probe is excited using a laser, and its fluorescence (R and G) at each element detected with a scanning confocal microscopeThe ratio between the signals in two channels (R:G) is calculated for each array spotRatios of intensity of Cy5/Cy3 probes is a reliable measure of the abundance of specific mRNA’s in each sample compared to “control”Oligo/cDNA Arrays MethodOligo/cDNA Arrays MethodTwoTwo--color color oligooligo/cDNA arrays/cDNA arraysmRNA from Sample 1 mRNA from Sample 2Scan and quantitate gene expression levelsTwoTwo--color color oligooligo/cDNA arrays/cDNA arraysAffymetrix® Affymetrix® GeneChipGeneChiparray formatarray format• One-color chip (biotin labeled cRNA, phycoerythrin-streptavidindetection)• Oligonucleotide probes are synthesized in situ on the chip• Semiconductor photolithography technology is used to synthesize oligos in situ on a glass substrate 1 cm2• Masking technology is used to build up oligonuclotides• Oligonucleotides corresponding to 5’, middle and 3’ sections of a gene of interest are used• Oligonucleotides corresponding to a perfect match and single mismatch are used to separate signal from noise• Hybridization is measured with a laser, quantified and stored asa raw value for comparison to data from another chipOligonucleotide “one color”