Toxicogenomics Toxicogenomics Toxicogenomics 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.mov http://video.niehs.nih.gov:7075/ramgen/ntc/ntc-eng03.rm http://www.niehs.nih.gov/nct/home.htm Gene 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) Microarray Experiments: Microarray Experiments: analysis of gene expression analysis 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 genes
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ToxicogenomicsToxicogenomics
Toxicogenomics is a new scientific field that elucidates how the entire genome is involved in biological responses of organisms exposed to environmental toxicants/stressors
• 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)
Microarray 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/discoveryDivide tumors into sub-classesDetermine effects of treatment
• Genome-wide (e.g., yeast)• Partial selection of known/unknown genes
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Scientific Areas in ToxicogenomicsScientific Areas in Toxicogenomics
Disease 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.
2. 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 data
DNA Microarray TechnologyDNA Microarray Technology
DNA sequences complementary to genes of interest are generated and laid out in
microscopic quantities on solid surfaces at defined positions
Presence of bound DNA is detected by fluorescence following laser excitation
and data is interrogated using specialized computational approaches
cDNAs (from mRNA) from samples are labeled with fluorescent probes and eluted over the
surface complementary DNA binds
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Spotted Array ConstructionFabrication of twoFabrication of two--color color oligooligo/cDNA arrays/cDNA arrays
Oligos (20-70 bp)or cDNAs 96-well plate 384-well plate printed on a glass slide
Two 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 microscope
The ratio between the signals in two channels (R:G) is calculated for each array spot
Ratios 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 Method
150 µm200 µm
TwoTwo--color color oligooligo/cDNA arrays/cDNA arrays
mRNA from Sample 1 mRNA from Sample 2
Scan and quantitate gene expression levels
TwoTwo--color color oligooligo/cDNA arrays/cDNA arrays
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AffymetrixAffymetrix®® GeneChipGeneChip array formatarray format
Affymetrix Affymetrix GeneChipGeneChip detection principledetection principle16-25 mer oligos, perfect match/mismatch, 8-20 per gene of interest
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Fluidics station(stain/wash)
~2 ½
in
~1 in
Scanner Analysis Software
Affymetrix Affymetrix GeneChipsGeneChips ““in your labin your lab””
Yeast Genome S98 Array Test3 Array Rat Toxicology U34 Array Rat Neurobiology U34 Array Rat Genome U34 Set P. aeruginosa Genome ArrayMurine Genome U74v2 Set Mouse Expression Set 430Human Genome U95 SetHuman Genome U133 SetHuman Genome Focus ArrayE. coli Antisense Genome ArrayDrosophila Genome ArrayC. elegans Genome ArrayArabidopsis ATH1 Genome ArrayCatalog Arrays
p53 AssayHuSNP Mapping AssayGenFlex Tag ArrayCYP450 AssayDNA Analysis Arrays
Made to Order ArraysCustomExpress™ Premier ArraysCustomExpress™ Advantage ArraysCustomExpress™ Arrays
Cost: $250-$1000 apiece
"Mainstream" arrays:"Mainstream" arrays:
Oligonucleotide based (two-color):Home-made vs. Agilent®Capacity: up to 25,000 genes/targets
Oligonucleotide based (one-color):Affymetrix®Capacity: up to 400,000 targets
Need for much greater number of targets on a single array:• minimize energy and materials needed for array production/processing• faster/cheaper• wider dynamic range, increased selectivity and sensitivity
Other platforms that receive attention:• Fiberoptic microarrays• Electrically addressable arrays• Electrokinetic microarrays
• Beads individually manufactured and QC’d with full length Oligator® oligos
• Pools produced containing equal mix of 1624 to 24,000+ bead types
• Bead pools applied to fiber bundle or BeadChip, and beads randomly self-assemble into microwells to form functional array with ~30 copies of each bead type
• All elements of the array quality checked for bead presence and type prior to supply (Gunderson et al. Decoding randomly ordered DNA arrays. Genome Research, May 2004)
Bioelectronic detection proceeds via a sandwich hybridization assay, wherein three critical components (capture probe, target, and signaling probe) are each present in the cartridge. The signaling probe serves to label the target upon hybridization. Electrons flow to the electrode surface only when the target is present and specifically hybridized to both signaling probe and capture probe. The current generated by this system is measured and interpreted by the eSensor™ DNA Detection Reader and Software.