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 nct home htm http www niehs nih gov multimedia qt ntc ntcaltcaption mov http video niehs nih gov 7075 ramgen ntc ntc eng03 rm 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 analysis of gene expression Genome wide e g yeast Partial selection of known unknown genes 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 Scientific 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 Classical Microarray Experiments Normal vs Disease Example Analysis of expression patterns in cancer Pattern of gene expression networks Novel gene association discovery Molecular Classification Example Comparison of Breast Tumors Samples classified into subtypes Genome Wide Analysis Example Genome wide expression in S cerevisiae Discovery of yet unidentified molecular pathways Microarray Technique Microarray A substrate with bound capture probes Capture probe An oligo cDNA with gene DNA sequence of interest Generic experimental steps 1 Fabrication Photolithography Affymetrix one color array 40 000 genes Printing Agilent two color arrays 45 000 genes Spotting In house two color arrays 24 000 genes 2 Target Generation from a sample of interest One color biotin labeled cRNA phycoerythrin streptavidin detection Two color Cy3 and Cy5 cDNA labeling 3 Hybridization 4 Analysis Scanning of array Amount of hybridized target is assessed Statistical interrogation of the data Fabrication DNA sequences complementary to genes of interest are generated and laid out in microscopic quantities on solid surfaces at defined positions Target generation and hybridization cDNAs from mRNA from samples are labeled with fluorescent probes and eluted over the surface complementary DNA binds Analysis DNA Microarray Technology Presence of bound DNA is detected by fluorescence following laser excitation and data is interrogated using specialized computational approaches Fabrication of Spotted two color oligo cDNA arrays Array Construction Oligos 20 70 bp or cDNAs 96 well plate 384 well plate printed on a glass slide Oligo cDNA Arrays Method 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 Two color oligo cDNA arrays 200 m 150 m Two color oligo cDNA arrays mRNA from Sample 1 mRNA from Sample 2 Scan and quantitate gene expression levels Affymetrix GeneChip array format One color chip biotin labeled cRNA phycoerythrin streptavidin detection 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 as a raw value for comparison to data from another chip Target preparation Array preparation Oligonucleotide one color Arrays Affymetrix mRNA reference sequences Perfect Match Mismatch probe sets in situ synthesis by photolitography One Gene Biotin labeled cRNA Double stranded cDNA Total mRNA Cells Tissue samples Ratio array1 array2 Affymetrix GeneChip detection principle 16 25 mer oligos perfect match mismatch 8 20 per gene of interest target mRNA sequence DNA probe pairs reference sequence perfect match oligo mismatch oligo perfect match probe cells Fluorescence Intensity Image mismatch probe cells Affymetrix GeneChips in your lab 1 2 in Catalog Arrays in Fluidics station stain wash Arabidopsis ATH1 Genome Array C elegans Genome Array Drosophila Genome Array E coli Antisense Genome Array Human Genome Focus Array Human Genome U133 Set Human Genome U95 Set Mouse Expression Set 430 Murine Genome U74v2 Set P aeruginosa Genome Array Rat Genome U34 Set Rat Neurobiology U34 Array Rat Toxicology U34 Array Test3 Array Yeast Genome S98 Array Scanner CustomExpress Arrays CustomExpress Advantage Arrays